copy 2013 Published by The Company of Biologists Ltd
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Growth hormone transgenesis and polyploidy increase metabolic rate alter 1
the cardiorespiratory response and influence HSP expression to acute 2
hypoxia in Atlantic salmon (Salmo salar) yolk-sac alevins 3
4
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Polymeropoulos E T 1 Plouffe D 2 LeBlanc S3 Elliott NG1 Currie S3 Frappell PB4 7
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9 1CSIRO National Food Futures Flagship CSIRO Marine and Atmospheric Research GPO Box 1538 Hobart Tasmania 10
7001 Australia 2AquaBounty Farms Souris Prince Edward Island C0A 2B0 Canada3Department of Biology Mount 11
Allison University Sackville NB E4L 1G7 Canada 4University of Tasmania 7001 Hobart Australia 12
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To whom correspondence should be addressed Elias Polymeropoulos CSIRO National Food Futures Flagship CSIRO 28
Marine and Atmospheric Research Marine Laboratories Castray Esplanade 7000 Hobart Australia Email 29
eliaspolymeropouloscsiroau Phone +61-3-6232-5208 30
httpjebbiologistsorglookupdoi101242jeb098913Access the most recent version at J Exp Biol Advance Online Articles First posted online on 27 March 2014 as doi101242jeb098913httpjebbiologistsorglookupdoi101242jeb098913Access the most recent version at
First posted online on 27 March 2014 as 101242jeb098913
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Summary 31
Growth hormone (GH) transgenic Atlantic salmon display accelerated growth rates compared 32
to non-transgenics GH-transgenic fish also display cardiorespiratory and metabolic 33
modifications that accompany the increased growth rate An elevated routine metabolic rate 34
has been described for pre- and post-smolt GH-transgenic salmon that also display 35
improvements in oxygen delivery to support the increased aerobic demand The early 36
ontogenic effects of GH-transgenesis on the respiratory and cellular physiology of fish 37
especially during adverse environmental conditions and the effect of polyploidy are unclear 38
Here we investigated the effects of GH-transgenesis and polyploidy on metabolic heart and 39
ventilation rates and heat shock protein (HSP) levels after exposure to acute hypoxia in post-40
hatch Atlantic salmon yolk-sac alevins Metabolic rate decreased with decreasing partial 41
pressures of oxygen in all genotypes In normoxia triploid transgenics displayed the highest 42
mass specific metabolic rates in comparison to diploid transgenics and non-transgenic 43
triploids which in contrast had higher rates than diploid non-transgenics In hypoxia we 44
observed a lower mass-specific metabolic rate in diploid non-transgenics compared to all 45
other genotypes However no evidence for improved O2 uptake through heart or ventilation 46
rate was found Heart rate decreased in diploid non-transgenics while ventilation rate 47
decreased in both diploid non-transgenics and triploid transgenics in severe hypoxia 48
Regardless of genotype or treatment inducible HSP70 was not expressed in alevins 49
Following hypoxia the constitutive isoform of HSP70 HSC70 decreased in transgenics and 50
HSP90 expression decreased in all genotypes These data suggest that physiological changes 51
through GH-transgenesis and polyploidy are manifested during early ontogeny in Atlantic 52
salmon 53
54
55
56
57
58
59
60
Key words Salmo salar hypoxia growth hormone transgenesis polyploidy metabolic rate 61
oxygen transport heat shock proteins 62
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Abbreviations 63
2n = diploid 64
3n = triploid 65
bpm = beats per minute 66
βwO2 = solubility of oxygen in water 67
fH = heart rate 68
fV = ventilation rate 69
GH = growth hormone 70
HSP = heat shock protein 71
kPa = kilopascal 72
M O2 = metabolic rate 73
M O2g = mass specific metabolic rate 74
PO2 = partial pressure of oxygen 75
Ta = ambient temperature 76
Tx = transgenic 77
Nt = non transgenic 78
79
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Introduction 80
Growth hormone (GH) transgenic fish have gained increasing attention in the aquaculture 81
industry as a result of accelerated growth rates (~two to tenfold) when compared to non-82
transgenic conspecifics (Devlin et al 1994 Martinez et al 1999 Shao Jun et al 1992 83
Stokstad 2002) Physiologically faster growth entails a higher demand for O2 through an 84
increase in metabolism In salmon from the parr stage onwards an increase in resting 85
routine metabolic rate has been verified in GH-transgenic fish (Cook et al 2000 Lee et al 86
2003 Stevens et al 1998) In association with an increased O2 demand modifications to the 87
cardiorespiratory physiology in GH-transgenic Atlantic salmon (Salmo salar) have been 88
documented that include an increase in heart size cardiac output hemoglobin concentration 89
muscle aerobic enzyme activity (Deitch et al 2006) gill surface area (Stevens and Sutterlin 90
1999) and modifications to cardiorespiratory function (Blier et al 2002 Cogswell et al 91
2002) 92
93
Due to the concerns of non-native transgenes impacting wild fish stocks in the event of an 94
escape from commercial aquaculture it has been suggested that transgenic fish species 95
should be reproductively sterile In some fish species including salmonids polyploidy 96
sporadically occurs as a natural evolutionary trait (Allendorf and Thorgaard 1984 Ferris 97
1984 Schultz 1980) and is often artificially induced to produce commercial triploid 98
reproductively sterile progeny The biological effects associated with triploidy in fish have in 99
part been investigated and are mainly attributed to heterozygosity cell size or gonadal 100
development (Benfey 1999 Maxime 2008) As a consequence of the additional complement 101
of chromosomes in triploid organisms nuclear size is increased in the cells of most organs 102
and tissues which in turn causes an increase in cell volume with overall body size being 103
maintained through hypoplasia in triploids (Benfey 1999) In theory sterile triploid 104
organisms have the potential for greater growth due to the abundance of genetic material as 105
well as the diversion of energy from gonadal growth to somatic growth during sexual 106
maturation In practice the evidence supporting increased growth rates in triploid organisms 107
is inconclusive (Tiwary et al 2004) since both slower (Galbreath et al 1994) as well as 108
equivalent growth rates have been observed in triploid populations relative to that of diploid 109
conspecifics (McGeachy et al 1995 Sacobie et al 2012) In the past poor performance of 110
triploid fish in comparison to diploids especially under conditions of high O2 demand has 111
been suggested to be related to changes to their respiratory physiology and therefore hypoxia 112
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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113
deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114
et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115
perform as well as diploid siblings Differences in hematological parameters associated with 116
triploidy as well as a reduction in gill surface area and metabolism have been reported for 117
salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118
understood Triploid Atlantic salmon have been documented to display a reduction in O2 119
carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120
O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121
systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122
brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123
counterparts (Stillwell and Benfey 1996) 124
125
Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126
normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127
Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128
contained within the yolk-sac Hence both O2 supply and demand may be under different 129
regulatory control compared to later stages of development or adulthood and may be 130
differentially affected by ploidal level or GH-transgenesis 131
132
Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133
significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134
the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135
exacerbated under adverse environmental conditions In order to cope with the potential 136
damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137
evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138
shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139
characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140
(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141
HSPs throughout ontogeny has gained increased attention and it has been suggested that 142
HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143
Expression of genes encoding HSPs show spatial and temporal specificity during early 144
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
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67 632
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Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
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Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
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Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
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heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
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Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
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679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
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Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
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699
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paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
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705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
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measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
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phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
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with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
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743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
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747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
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Physiol A Mol Integr Physiol 87 157-160 750
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
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to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
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Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
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Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
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transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
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shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
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787
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Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
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Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
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Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Jour
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
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2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
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Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Summary 31
Growth hormone (GH) transgenic Atlantic salmon display accelerated growth rates compared 32
to non-transgenics GH-transgenic fish also display cardiorespiratory and metabolic 33
modifications that accompany the increased growth rate An elevated routine metabolic rate 34
has been described for pre- and post-smolt GH-transgenic salmon that also display 35
improvements in oxygen delivery to support the increased aerobic demand The early 36
ontogenic effects of GH-transgenesis on the respiratory and cellular physiology of fish 37
especially during adverse environmental conditions and the effect of polyploidy are unclear 38
Here we investigated the effects of GH-transgenesis and polyploidy on metabolic heart and 39
ventilation rates and heat shock protein (HSP) levels after exposure to acute hypoxia in post-40
hatch Atlantic salmon yolk-sac alevins Metabolic rate decreased with decreasing partial 41
pressures of oxygen in all genotypes In normoxia triploid transgenics displayed the highest 42
mass specific metabolic rates in comparison to diploid transgenics and non-transgenic 43
triploids which in contrast had higher rates than diploid non-transgenics In hypoxia we 44
observed a lower mass-specific metabolic rate in diploid non-transgenics compared to all 45
other genotypes However no evidence for improved O2 uptake through heart or ventilation 46
rate was found Heart rate decreased in diploid non-transgenics while ventilation rate 47
decreased in both diploid non-transgenics and triploid transgenics in severe hypoxia 48
Regardless of genotype or treatment inducible HSP70 was not expressed in alevins 49
Following hypoxia the constitutive isoform of HSP70 HSC70 decreased in transgenics and 50
HSP90 expression decreased in all genotypes These data suggest that physiological changes 51
through GH-transgenesis and polyploidy are manifested during early ontogeny in Atlantic 52
salmon 53
54
55
56
57
58
59
60
Key words Salmo salar hypoxia growth hormone transgenesis polyploidy metabolic rate 61
oxygen transport heat shock proteins 62
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Abbreviations 63
2n = diploid 64
3n = triploid 65
bpm = beats per minute 66
βwO2 = solubility of oxygen in water 67
fH = heart rate 68
fV = ventilation rate 69
GH = growth hormone 70
HSP = heat shock protein 71
kPa = kilopascal 72
M O2 = metabolic rate 73
M O2g = mass specific metabolic rate 74
PO2 = partial pressure of oxygen 75
Ta = ambient temperature 76
Tx = transgenic 77
Nt = non transgenic 78
79
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Introduction 80
Growth hormone (GH) transgenic fish have gained increasing attention in the aquaculture 81
industry as a result of accelerated growth rates (~two to tenfold) when compared to non-82
transgenic conspecifics (Devlin et al 1994 Martinez et al 1999 Shao Jun et al 1992 83
Stokstad 2002) Physiologically faster growth entails a higher demand for O2 through an 84
increase in metabolism In salmon from the parr stage onwards an increase in resting 85
routine metabolic rate has been verified in GH-transgenic fish (Cook et al 2000 Lee et al 86
2003 Stevens et al 1998) In association with an increased O2 demand modifications to the 87
cardiorespiratory physiology in GH-transgenic Atlantic salmon (Salmo salar) have been 88
documented that include an increase in heart size cardiac output hemoglobin concentration 89
muscle aerobic enzyme activity (Deitch et al 2006) gill surface area (Stevens and Sutterlin 90
1999) and modifications to cardiorespiratory function (Blier et al 2002 Cogswell et al 91
2002) 92
93
Due to the concerns of non-native transgenes impacting wild fish stocks in the event of an 94
escape from commercial aquaculture it has been suggested that transgenic fish species 95
should be reproductively sterile In some fish species including salmonids polyploidy 96
sporadically occurs as a natural evolutionary trait (Allendorf and Thorgaard 1984 Ferris 97
1984 Schultz 1980) and is often artificially induced to produce commercial triploid 98
reproductively sterile progeny The biological effects associated with triploidy in fish have in 99
part been investigated and are mainly attributed to heterozygosity cell size or gonadal 100
development (Benfey 1999 Maxime 2008) As a consequence of the additional complement 101
of chromosomes in triploid organisms nuclear size is increased in the cells of most organs 102
and tissues which in turn causes an increase in cell volume with overall body size being 103
maintained through hypoplasia in triploids (Benfey 1999) In theory sterile triploid 104
organisms have the potential for greater growth due to the abundance of genetic material as 105
well as the diversion of energy from gonadal growth to somatic growth during sexual 106
maturation In practice the evidence supporting increased growth rates in triploid organisms 107
is inconclusive (Tiwary et al 2004) since both slower (Galbreath et al 1994) as well as 108
equivalent growth rates have been observed in triploid populations relative to that of diploid 109
conspecifics (McGeachy et al 1995 Sacobie et al 2012) In the past poor performance of 110
triploid fish in comparison to diploids especially under conditions of high O2 demand has 111
been suggested to be related to changes to their respiratory physiology and therefore hypoxia 112
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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113
deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114
et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115
perform as well as diploid siblings Differences in hematological parameters associated with 116
triploidy as well as a reduction in gill surface area and metabolism have been reported for 117
salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118
understood Triploid Atlantic salmon have been documented to display a reduction in O2 119
carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120
O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121
systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122
brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123
counterparts (Stillwell and Benfey 1996) 124
125
Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126
normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127
Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128
contained within the yolk-sac Hence both O2 supply and demand may be under different 129
regulatory control compared to later stages of development or adulthood and may be 130
differentially affected by ploidal level or GH-transgenesis 131
132
Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133
significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134
the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135
exacerbated under adverse environmental conditions In order to cope with the potential 136
damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137
evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138
shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139
characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140
(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141
HSPs throughout ontogeny has gained increased attention and it has been suggested that 142
HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143
Expression of genes encoding HSPs show spatial and temporal specificity during early 144
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
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Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Abbreviations 63
2n = diploid 64
3n = triploid 65
bpm = beats per minute 66
βwO2 = solubility of oxygen in water 67
fH = heart rate 68
fV = ventilation rate 69
GH = growth hormone 70
HSP = heat shock protein 71
kPa = kilopascal 72
M O2 = metabolic rate 73
M O2g = mass specific metabolic rate 74
PO2 = partial pressure of oxygen 75
Ta = ambient temperature 76
Tx = transgenic 77
Nt = non transgenic 78
79
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Introduction 80
Growth hormone (GH) transgenic fish have gained increasing attention in the aquaculture 81
industry as a result of accelerated growth rates (~two to tenfold) when compared to non-82
transgenic conspecifics (Devlin et al 1994 Martinez et al 1999 Shao Jun et al 1992 83
Stokstad 2002) Physiologically faster growth entails a higher demand for O2 through an 84
increase in metabolism In salmon from the parr stage onwards an increase in resting 85
routine metabolic rate has been verified in GH-transgenic fish (Cook et al 2000 Lee et al 86
2003 Stevens et al 1998) In association with an increased O2 demand modifications to the 87
cardiorespiratory physiology in GH-transgenic Atlantic salmon (Salmo salar) have been 88
documented that include an increase in heart size cardiac output hemoglobin concentration 89
muscle aerobic enzyme activity (Deitch et al 2006) gill surface area (Stevens and Sutterlin 90
1999) and modifications to cardiorespiratory function (Blier et al 2002 Cogswell et al 91
2002) 92
93
Due to the concerns of non-native transgenes impacting wild fish stocks in the event of an 94
escape from commercial aquaculture it has been suggested that transgenic fish species 95
should be reproductively sterile In some fish species including salmonids polyploidy 96
sporadically occurs as a natural evolutionary trait (Allendorf and Thorgaard 1984 Ferris 97
1984 Schultz 1980) and is often artificially induced to produce commercial triploid 98
reproductively sterile progeny The biological effects associated with triploidy in fish have in 99
part been investigated and are mainly attributed to heterozygosity cell size or gonadal 100
development (Benfey 1999 Maxime 2008) As a consequence of the additional complement 101
of chromosomes in triploid organisms nuclear size is increased in the cells of most organs 102
and tissues which in turn causes an increase in cell volume with overall body size being 103
maintained through hypoplasia in triploids (Benfey 1999) In theory sterile triploid 104
organisms have the potential for greater growth due to the abundance of genetic material as 105
well as the diversion of energy from gonadal growth to somatic growth during sexual 106
maturation In practice the evidence supporting increased growth rates in triploid organisms 107
is inconclusive (Tiwary et al 2004) since both slower (Galbreath et al 1994) as well as 108
equivalent growth rates have been observed in triploid populations relative to that of diploid 109
conspecifics (McGeachy et al 1995 Sacobie et al 2012) In the past poor performance of 110
triploid fish in comparison to diploids especially under conditions of high O2 demand has 111
been suggested to be related to changes to their respiratory physiology and therefore hypoxia 112
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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113
deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114
et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115
perform as well as diploid siblings Differences in hematological parameters associated with 116
triploidy as well as a reduction in gill surface area and metabolism have been reported for 117
salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118
understood Triploid Atlantic salmon have been documented to display a reduction in O2 119
carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120
O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121
systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122
brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123
counterparts (Stillwell and Benfey 1996) 124
125
Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126
normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127
Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128
contained within the yolk-sac Hence both O2 supply and demand may be under different 129
regulatory control compared to later stages of development or adulthood and may be 130
differentially affected by ploidal level or GH-transgenesis 131
132
Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133
significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134
the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135
exacerbated under adverse environmental conditions In order to cope with the potential 136
damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137
evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138
shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139
characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140
(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141
HSPs throughout ontogeny has gained increased attention and it has been suggested that 142
HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143
Expression of genes encoding HSPs show spatial and temporal specificity during early 144
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
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Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
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619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
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Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
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Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
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McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
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OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
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Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
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Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
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Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
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Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Introduction 80
Growth hormone (GH) transgenic fish have gained increasing attention in the aquaculture 81
industry as a result of accelerated growth rates (~two to tenfold) when compared to non-82
transgenic conspecifics (Devlin et al 1994 Martinez et al 1999 Shao Jun et al 1992 83
Stokstad 2002) Physiologically faster growth entails a higher demand for O2 through an 84
increase in metabolism In salmon from the parr stage onwards an increase in resting 85
routine metabolic rate has been verified in GH-transgenic fish (Cook et al 2000 Lee et al 86
2003 Stevens et al 1998) In association with an increased O2 demand modifications to the 87
cardiorespiratory physiology in GH-transgenic Atlantic salmon (Salmo salar) have been 88
documented that include an increase in heart size cardiac output hemoglobin concentration 89
muscle aerobic enzyme activity (Deitch et al 2006) gill surface area (Stevens and Sutterlin 90
1999) and modifications to cardiorespiratory function (Blier et al 2002 Cogswell et al 91
2002) 92
93
Due to the concerns of non-native transgenes impacting wild fish stocks in the event of an 94
escape from commercial aquaculture it has been suggested that transgenic fish species 95
should be reproductively sterile In some fish species including salmonids polyploidy 96
sporadically occurs as a natural evolutionary trait (Allendorf and Thorgaard 1984 Ferris 97
1984 Schultz 1980) and is often artificially induced to produce commercial triploid 98
reproductively sterile progeny The biological effects associated with triploidy in fish have in 99
part been investigated and are mainly attributed to heterozygosity cell size or gonadal 100
development (Benfey 1999 Maxime 2008) As a consequence of the additional complement 101
of chromosomes in triploid organisms nuclear size is increased in the cells of most organs 102
and tissues which in turn causes an increase in cell volume with overall body size being 103
maintained through hypoplasia in triploids (Benfey 1999) In theory sterile triploid 104
organisms have the potential for greater growth due to the abundance of genetic material as 105
well as the diversion of energy from gonadal growth to somatic growth during sexual 106
maturation In practice the evidence supporting increased growth rates in triploid organisms 107
is inconclusive (Tiwary et al 2004) since both slower (Galbreath et al 1994) as well as 108
equivalent growth rates have been observed in triploid populations relative to that of diploid 109
conspecifics (McGeachy et al 1995 Sacobie et al 2012) In the past poor performance of 110
triploid fish in comparison to diploids especially under conditions of high O2 demand has 111
been suggested to be related to changes to their respiratory physiology and therefore hypoxia 112
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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113
deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114
et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115
perform as well as diploid siblings Differences in hematological parameters associated with 116
triploidy as well as a reduction in gill surface area and metabolism have been reported for 117
salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118
understood Triploid Atlantic salmon have been documented to display a reduction in O2 119
carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120
O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121
systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122
brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123
counterparts (Stillwell and Benfey 1996) 124
125
Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126
normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127
Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128
contained within the yolk-sac Hence both O2 supply and demand may be under different 129
regulatory control compared to later stages of development or adulthood and may be 130
differentially affected by ploidal level or GH-transgenesis 131
132
Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133
significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134
the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135
exacerbated under adverse environmental conditions In order to cope with the potential 136
damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137
evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138
shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139
characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140
(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141
HSPs throughout ontogeny has gained increased attention and it has been suggested that 142
HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143
Expression of genes encoding HSPs show spatial and temporal specificity during early 144
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
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Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
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Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
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Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113
deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114
et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115
perform as well as diploid siblings Differences in hematological parameters associated with 116
triploidy as well as a reduction in gill surface area and metabolism have been reported for 117
salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118
understood Triploid Atlantic salmon have been documented to display a reduction in O2 119
carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120
O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121
systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122
brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123
counterparts (Stillwell and Benfey 1996) 124
125
Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126
normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127
Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128
contained within the yolk-sac Hence both O2 supply and demand may be under different 129
regulatory control compared to later stages of development or adulthood and may be 130
differentially affected by ploidal level or GH-transgenesis 131
132
Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133
significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134
the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135
exacerbated under adverse environmental conditions In order to cope with the potential 136
damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137
evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138
shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139
characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140
(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141
HSPs throughout ontogeny has gained increased attention and it has been suggested that 142
HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143
Expression of genes encoding HSPs show spatial and temporal specificity during early 144
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145
required for normal tissue development (Krone et al 2003) 146
147
Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148
cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149
alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150
changes to respiration will affect the heat shock response under conditions of low O2 151
availability The cellular stress response to low environmental O2 levels is variable in fish and 152
high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153
have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154
2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155
tissue of sparids (family Sparidae) has been reported after exogenous administration of 156
growth hormone (Deane et al 1999) Despite this body of research we know very little 157
regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158
159
In the present study we tested for differences in metabolic rate cardiorespiratory function 160
and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161
transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162
these parameters are altered under hypoxic conditions It was hypothesised that GH-163
transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164
uptake at this early developmental stage This increase may be supported by improved O2 165
delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166
Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167
within) to hypoxia there is no clear evidence that this is the case in the present study 168
Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169
of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170
stress response that may be augmented by increased anabolic processes or an increased 171
metabolism associated with GH-transgenesis 172
173
174
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
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Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
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Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Results 175
176
Treatments and Measurements 177
Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178
(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179
alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180
were conducted of MO2
heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181
specific M O2 was calculated on the basis of yolk-free wet body mass 182
183
Additionally we tested the cellular stress response of alevins (see Series III) of the different 184
genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185
of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186
alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187
(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188
Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189
experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190
alevins per genotype were maintained under indentical experimental conditions in normoxia 191
(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192
described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193
mesh separating the alevins from escaping into the surrounding water The control group was 194
maintained within the same multiwell plate for a duration that matched that of the 195
experimental animals (measurement + 24 h) this included a quick removal from the wells to 196
mimic the handling stress to which the experimental animals were exposed After the 197
required interval specimens were taken from the chambers kept in ice water measured for 198
length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199
performed 200
201
Body mass and morphometry 202
Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203
genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
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67 632
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Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
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Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
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Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
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heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
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Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
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smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
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Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
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705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
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measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
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phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
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McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
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731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
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743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
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Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
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Physiol A Mol Integr Physiol 87 157-160 750
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
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to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
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Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
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shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
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Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
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Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
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Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Jour
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
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2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
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Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205
length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206
did not differ statistically in any of the parameters measured in animals used in Series I 207
208
Series I Mass-specific metabolic rate 209
Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210
pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211
constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212
sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213
(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214
hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215
3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216
comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217
hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218
3nTx alevins were not statistically different from each other (Fig 2 inset) 219
220
Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221
ventilation rate 222
The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223
(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224
ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225
normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226
previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227
levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228
overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229
Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230
16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231
2nNt alevins 232
233
3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234
fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
The
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Jour
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f Exp
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enta
l Bio
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ndash A
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D A
UTH
OR
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NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Jour
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24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
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l Bio
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27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236
in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237
3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238
normoxic and intermediate hypoxic conditions no difference in fV was detected between 239
genotypes 240
241
Series III HSP levels 242
The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243
incorporated within Series I results Western blot analysis of whole animal samples revealed 244
that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245
contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246
relative expression of HSC70 between genotypes were observed in the control samples kept 247
in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248
transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249
relative HSP90 expression in control groups were similar but significantly lower in all 250
hypoxia treated alevins (Fig 4b P lt 0001) 251
252
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Jour
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Discussion 253
In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254
metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255
present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256
metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257
transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258
metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259
separate methodogical approach while simultaneously measuring heart rate and ventilation 260
rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261
Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262
in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263
physiological mechanisms to ensure O2 delivery These might include morphological changes 264
such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265
concentration or changes in cardiac stroke volume The difference in metabolic rate among 266
groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267
under control conditions and hypoxia did not appear to elicit a cellular stress response 268
However hypoxia resulted in differential decreased HSP levels among groups with 269
transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270
decreased under hypoxic conditions across all genotypes 271
272
Series I and Series II 273
The M O2 curves in alevins that were measured in Series I using the closed system approach 274
generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275
14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276
intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277
This pattern was unexpected as it does not fully comply with either classic oxygen 278
conformity or regulation curves for M O2 (see Richards 2009) 279
280
It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281
unconstrained alevin at the beginning of the measurement where the alevins have not fully 282
settled or reached resting M O2 after handling However once the animals were put in the 283
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
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67 632
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Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
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Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
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Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
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heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
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Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
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Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
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Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
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679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
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Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
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Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
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Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
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paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
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Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
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consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
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measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
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phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
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with diploid fish Fish Fish 9 67-78 726
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McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
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731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
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OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
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to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
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Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
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Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
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transfer Aquaculture 334-337 58-64 778
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and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
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Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
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against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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Jour
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
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2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
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Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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respirometry chambers they settled on the mesh within a few minutes and did not move 284
(personal observation) In addition when M O2 was measured in the open flow respirometry 285
system and heart rate and ventilation rates where measured simultaneously it was verified 286
that resting heart and ventilation rates were established at a minimum usually within ~30 287
min The period of the initial steep decline in the closed system is significantly longer (~90-288
120 min) and therefore unlikely to be related to activity 289
290
The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291
explain In section II the alevins show a greater ability to maintain M O2 which could be 292
indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293
specific PO2 This pathway might involve the reallocation of ATP to different ATP 294
consuming pathways (ie protein synthesis degradation ion transport) that are more 295
critically involved in the hypoxic metabolic response During the exposure to a gradually 296
decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297
ATP production that support the maintenance of M O2 Even though the alevins appear to 298
settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299
movement) during an experiment is likely to add further insight into the M O2 pattern 300
observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301
activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302
further insight into changes in metabolic pathways Another simple explanation for the 303
triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304
state of the animal section II might then correspond to the true settled state The decrease in 305
M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306
further reduce metabolic rate as part of ldquometabolic depressionrdquo 307
In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308
depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309
Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310
availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311
lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312
(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313
diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314
the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
The
Jour
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f Exp
erim
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ndash A
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UTH
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IPT
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
The
Jour
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l Bio
logy
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UTH
OR
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24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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Jour
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EPTE
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Jour
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26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
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enta
l Bio
logy
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CC
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27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316
in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317
an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318
in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319
chicken embryo (Mortola et al 2009) 320
321
The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322
convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323
ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324
alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325
the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326
cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327
feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328
this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329
shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330
al 2011) 331
332
In the present study we observed bradycardia and a decrease in ventilation in response to 333
severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334
this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335
This result implies that although there was no general coupling of fH or fV to M O2 the 336
chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337
increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338
339
The differences in M O2g in comparison between Series I and Series II are most likely due to 340
the different experimental designs where the relatively greater constraint of the animal in 341
Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342
with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343
more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344
trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345
indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
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developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
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Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
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Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
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physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
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Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
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heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
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Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
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Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
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Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
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705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
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743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
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747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
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Physiol A Mol Integr Physiol 87 157-160 750
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Jour
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775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
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Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
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Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
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Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
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2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
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Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347
2nNt) 348
349
If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350
between genotypes of similar body mass the same response in fH and fV could be expected 351
As this is not the case the animal must be using physiological strategies to maximize O2 352
uptake These may include improved O2 extraction capabilities via an increase in cardiac 353
output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354
volume or an increased hemoglobin concentration Equally changes to the diffusive 355
properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356
cardiorespiratory response These possibilities require further experimentation 357
358
It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359
associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360
study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361
increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362
ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363
and triploidy on metabolic physiology which so far has not been investigated Whereas the 364
increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365
the light of a greater energy requirement for developmental and anabolic processes 366
alterations to respiratory mechanisms due to triploidy remain elusive It has been 367
hypothesised that the larger cell size of most tissues and the distinct morphology 368
characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369
therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370
(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371
distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372
established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373
accounts of alterations to hematological parameters in the literature in particular the size and 374
shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375
inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376
2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377
of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378
this question 379
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
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(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
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and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
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smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
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Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
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K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
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Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
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Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
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Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
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Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
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Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
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Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
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Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
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Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
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Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
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Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
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Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
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McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
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McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
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Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
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OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
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Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
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Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
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Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
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Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
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Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
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Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
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Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
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Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
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Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
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Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
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Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
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Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
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Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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380
Series III 381
On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382
to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383
(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384
hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385
of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386
Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387
hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388
rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389
in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390
mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391
increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392
western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393
(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394
2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395
hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396
studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397
tolerance protecting proteins from damage associated with low oxygen andor the sudden 398
return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399
to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400
spatial and species-specific regulation 401
402
Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403
induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404
is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405
to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406
(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407
stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408
indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409
obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Jour
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f Exp
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enta
l Bio
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ndash A
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UTH
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IPT
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Jour
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l Bio
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27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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of adult rainbow trout are able to mount an inducible heat shock response under severe 411
hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412
phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413
salmonids at least are not energetically limited in terms of their heat shock response Protein 414
synthesis in earlier developmental stages however may be more susceptible to these 415
energetic constraints 416
417
Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418
transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419
finding can not be explained by differences in metabolism Our knowledge of the cellular and 420
molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421
synthesis is preferentially directed towards muscle growth in transgenic animals over 422
constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423
Interestingly the differences in metabolic rate between groups was not reflected in 424
differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425
anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426
however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427
initial metabolic rate Future studies should examine the effects of long-term exposure to 428
hypoxia on the cellular stress response throughout development in transgenic animals to 429
improve our understanding of the involvement of HSPs in normal growth and development as 430
well as in hypoxia tolerance 431
432
Conclusions and perspectives 433
This is the first study examining the metabolic cardiorespiratory and cellular stress response 434
to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435
polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436
metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437
Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438
which may have a negative effect on the ability of the animal to deal with harsh 439
environments We see an important role in further investigating respiratory and cellular 440
responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441
and comparing them to juvenile and adult life stages It is likely that physiological differences 442
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Jour
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UTH
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IPT
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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UTH
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675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Jour
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erim
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l Bio
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27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
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Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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during early life stages are carried on to adulthood and might affect robustness or 443
susceptibility to environmental stress in an aquaculture environment 444
Materials and Methods 445
Experimental animals 446
Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447
~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448
reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449
(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450
UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451
diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452
single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453
stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454
(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455
hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456
after fertilisation which prevents the extrusion of the second polar body during meiotic 457
development and renders the animals sterile with an effectiveness of better than 99 458
459
Yolk-sac alevin mass and morphometry 460
Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461
(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462
following each experiment The total wet weight of each alevin was measured in grams to the 463
nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464
each alevin was measured in grams following the separation of the the yolk-sac from the 465
alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466
rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467
dissecting microscope 468
469
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
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Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
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Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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644
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physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
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78 347-355 651
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triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
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Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
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668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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IPT
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
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743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
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Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
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IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Series I Measurement of metabolic rate in alevins 470
The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471
fluorescence-based closed system respirometry Each alevin was placed in an individual well 472
in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473
Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474
of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475
a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476
the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477
animals for calibration purposes two were filled with normoxic water from the rearing trays 478
and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479
Each well was sealed with a glass cover slip using vacuum grease care being taken to 480
exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481
PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482
set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483
the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484
the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485
intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486
physical disturbance confirmed that all alevins were alive at the end of each experiment 487
Before every measurement optodes were calibrated by alternatively filling the wells with 1 488
sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489
water (PO2 = 21 kPa) 490
491
The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492
time in each well after taking into account the diffusive flux of O2 that occurs through the 493
polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494
495
Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496
body mass among individuals The volume of the chamber was corrected for the volume 497
displaced by each individual according to its total wet mass assuming a density of 1gml the 498
stainless steel ball bearing and mesh 499
500
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
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743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Jour
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27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
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28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501
individual alevins 502
Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503
were performed on individual alevins using a combined optophysiological and respirometry 504
system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505
chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506
temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507
temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508
plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509
through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510
Systems Instrumentation Burlington Vermont) the water first passing through a heat 511
exchanger in the water bath to maintain the required temperature 512
513
514
To minimise handling stress alevins were acclimated within the chamber under constant 515
flow of normoxic water for 1 h before measurement The chamber was then sealed for 516
approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517
within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518
and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519
achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520
gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521
(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522
pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523
sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524
surrounding the alevin 525
526
For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527
PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528
in series downstream to the respirometry chamber and maintained at the same ambient 529
temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530
O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531
2005) Following a period where the chamber was sealed it was then flushed with fresh 532
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
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Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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IPT
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
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Jour
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f Exp
erim
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l Bio
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D A
UTH
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IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533
kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534
mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535
compartment was sealed (equation 1) 536
537
M O2= βwO2middotPO2middotV
t (1) 538
539
Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540
541
The validation of the intermittantly sealed flow-through respirometry system is described in 542
detail in the Appendix 543
544
During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545
measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546
SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547
was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548
Video information was processed by a video dimension analyser (V94 Living Systems 549
Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550
card (Roxio 315-E) In principle the instrument operates on the relative optical density 551
changes of border structures (ie border of beating heart or moving operculum) and generates 552
a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553
ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554
within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555
sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556
measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557
fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558
operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559
response to physical disturbance confirmed that all alevins were alive at the end of each 560
experiment 561
562
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
The
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
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References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
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Jour
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f Exp
erim
enta
l Bio
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ndash A
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UTH
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NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
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UTH
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SCR
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24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
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25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
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Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
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Jour
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f Exp
erim
enta
l Bio
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CC
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D A
UTH
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NU
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IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
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erim
enta
l Bio
logy
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28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
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l Bio
logy
ndash A
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OR
MA
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29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
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logy
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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
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The
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Series III Heat shock protein (HSP) immunodetection 563
Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564
et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565
buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566
Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567
pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568
Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569
removed and stored at -80oC The soluble protein concentration of each sample was measured 570
using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571
572
To determine HSP levels immunodetection via western blot was performed using the Novex 573
Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574
4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575
system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576
(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577
shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578
between gels For immunodetection rabbit salmonid-specific primary antibodies against 579
HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580
concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581
HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582
HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583
isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584
Life Sciences) was used at a concentration of 150 000 as secondary antibody 585
586
Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587
Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588
ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589
density of the appropriate standard to obtain the relative band density After each round of 590
immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591
(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592
pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593
The
Jour
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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
The
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logy
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22
References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
The
Jour
nal o
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erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
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25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
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enta
l Bio
logy
ndash A
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EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
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enta
l Bio
logy
ndash A
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D A
UTH
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MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
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l Bio
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NU
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IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
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EPTE
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30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
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logy
ndash A
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EPTE
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IPT
The
Jour
nal o
f Exp
erim
enta
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logy
ndash A
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EPTE
D A
UTH
OR
MA
NU
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IPT
The
Jour
nal o
f Exp
erim
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l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
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IPT
The
Jour
nal o
f Exp
erim
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logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
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logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
21
43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594
using a different antibody 595
596
Statistical analysis 597
Morphometrical parameters were compared between genotypes using one-way ANOVA 598
Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599
were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600
2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601
HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602
transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603
comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604
605
606
Acknowledgements 607
Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608
Discovery Grants Harold Crabtree Foundation 609
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
22
References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
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enta
l Bio
logy
ndash A
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EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
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EPTE
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NU
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IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
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enta
l Bio
logy
ndash A
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EPTE
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UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
22
References 610
Airaksinen S and Nikinmaa M (1995) Effect of hemoglobin concentration on the 611
oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612
613
Allendorf F W and Thorgaard G H (1984) In Evolutionary Genetics of Fishes (ed B 614
J Turner) pp 1-53 Springer US 615
616
Aquabounty Technologies (2010) lsquoReport Prepared for the FDA Veterinary Medicine 617
Advisory Committeersquo 618
619
Barrionuevo W R and Burggren W W (1999) O2 consumption and heart rate in 620
developing zebrafish (Danio rerio) Influence of temperature and ambient O2 Am J Physiol 621
Regul Integr Comp Physiol 276 R505-R513 622
623
Barton B A (2002) Stress in fishes A diversity of responses with particular reference to 624
changes in circulating corticosteroids Integr Comp Biol 42 517-525 625
626
Basu N Todgham A E Ackerman P A Bibeau M R Nakano K Schulte P M 627
and Iwama G K (2002) Heat shock protein genes and their functional significance in fish 628
Gene 295 173-183 629
630
Benfey T J (1999) The Physiology and Behavior of Triploid Fishes Rev Fish Sci 7 39-631
67 632
633
Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634
digestive enzymes or metabolic capacity of the tissues Insight from transgenic coho salmon 635
Aquaculture 209 379-384 636
637
Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638
juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639
JFish Biol 71 1179-1191 640
641
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
23
Burggren W W (2004) What is the purpose of the embryonic heart beat Or how facts can 642
ultimately prevail over physiological dogma Physiol Biochem Zool 77 333-345 643
644
Burggren W W and Pinder A W (1991) Ontogeny of cardiovascular and respiratory 645
physiology in lower vertebrates Ann Rev Physiol 53 107-135 646
647
Clark T D Ryan T Ingram B A Woakes A J Butler P J and Frappell P B 648
(2005) Factorial aerobic scope is independent of temperature and primarily modulated by 649
heart rate in exercising Murray cod (Maccullochella peelii peelii) Physiol Biochem Zool 650
78 347-355 651
652
Cogswell A T Benfey T J and Sutterlin A M (2002) The hematology of diploid and 653
triploid transgenic Atlantic salmon (Salmo salar) Fish Physiol Biochem 24 271-277 654
655
Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656
oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon 657
(Salmo salar) Aquaculture 188 47-63 658
659
Currie S Bagatto B DeMille M Learner A LeBlanc D Marks C Ong K Parker 660
J Templeman N Tufts BL and Wright PA (2010) Metabolism nitrogen excretion and 661
heat shock proteins in the central mudminnow (Umbra limi) a faculatative air-breathing fish 662
living in a variable environment Can J Zool 8843-58 663
664
Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665
cardiomyocytes from the overwintering common frog Rana temporaria Physiol Biochem 666
Zool 74 420-428 667
668
Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669
(Oncorhynchus mykiss) red blood cells J Exp Biol 200 607-614 670
671
Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672
and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673
Endocrinol 161 413-421 674
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
24
675
Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676
W R and Gamperl A K (2006) Cardiorespiratory modifications and limitations in post-677
smolt growth hormone transgenic Atlantic salmon Salmo salar J Exp Biol 209 1310-1325 678
679
Dejours P (1981) Principles of Comparative Respiratory Physiology ElsevierNorth-680
Holland Biomedical Press Amsterdam-New York-Oxford 681
682
Devlin R H Yesaki T Y Biagi C A Donaldson E M Swanson P and Chan W 683
K (1994) Extraordinary salmon growth (7) Nature 371 209-210 684
685
Feder M E and Hofmann G E (1999) Heat-shock proteins molecular chaperones and 686
the stress response Evolutionary and ecological physiology Ann Rev Physiol 61 243-282 687
688
Ferris S D (1984) Tetraploidy and the evolution of the catostomid fishes In Evolutionary 689
Genetics of Fishes (ed B J Turner) pp 55-93 Springer US 690
691
Fu C Wilson J M Rombough P J and Brauner C J (2010) Ions first Na+ uptake 692
shifts from the skin to the gills before O2 uptake in developing rainbow trout Oncorhynchus 693
mykiss Proc R Soc B 277 1553-1560 694
695
Golding S Mohan R M Paterson D J (2000) Chronic intermittent hypoxia promotes 696
differential expression of stress proteins following in vitro hypoxia in guinea pig hearts 697
FASEB J 14(4) A586 698
699
Graham J B Dudley R Aguilar N M and Gans C (1995) Implications of the late 700
paleozoic oxygen pulse for physiology and evolution Nature 375 117-120 701
702
Holeton G F (1971) Respiratory and circulatory responses of rainbow trout larvae to 703
carbon monoxide and to hypoxia J Exp Biol 55 683-694 704
705
Kregel K C (2002) Heat shock proteins modifying factors in physiological stress 706
responses and acquired thermotolerance J Appl Physiol 92 2177-2186 707
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
25
708
LeBlanc S Hglund E Gilmour KM and Currie S (2012) Hormonal modulation of 709
the heat shock response Insights from fish with divergent cortisol stress responses Am J 710
Physiol Regul Integr Comp Physiol 302(1) 184-192 711
712
Lee C G Devlin R H and Farrell A P (2003) Swimming performance oxygen 713
consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-714
ranched coho salmon J Fish Biol 62 753-766 715
716
Lowry O H Rosebrough N J Farr A L and Randall R J (1951) Protein 717
measurement with the folin phenol reagent J Biol Chem 193 265-275 718
719
Martinez R Arenal A Estrada M P Herrera F Huerta V Vazquez J Sanchez 720
T and De La Fuente J (1999) Mendelian transmission transgene dosage and growth 721
phenotype in transgenic tilapia (Oreochromis homorum) showing ectopic expression of 722
homologous growth hormone Aquaculture 173 271-283 723
724
Maxime V (2008) The physiology of triploid fish Current knowledge and comparisons 725
with diploid fish Fish Fish 9 67-78 726
727
McDonald D G and McMahon B R (1977) Respiratory development in Arctic char 728
Salvelinus alpinus under conditions of normoxia and chronic hypoxia Can J Zool 55 1461-729
1467 730
731
McGeachy S A Benfey T J and Friars G W (1995) Freshwater performance of 732
triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture Aquaculture 137 733
333-341 734
735
Miller S C Gillis T E and Wright P A (2011) The ontogeny of regulatory control of 736
the rainbow trout (Oncorhynchus mykiss) heart and how this is influenced by chronic hypoxia 737
exposure J Exp Biol 214 2065-2072 738
739
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
26
Mortola J P Wills K Trippenbach T and Al Awam K (2009) Interactive effects of 740
temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken 741
embryo Comp Biochem Physiol A Mol Integr Physiol155 301-308 742
743
OFlynn F M McGeachy S A Friars G W Benfey T J and Bailey J K (1997) 744
Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L) ICES J Mar 745
Sci 54 1160-1165 746
747
Oliva-Teles A and Kaushik S J (1987) Nitrogen and energy metabolism during the early 748
ontogeny of diploid and triploid rainbow trout (Salmo gairdneri R) Comp Biochem 749
Physiol A Mol Integr Physiol 87 157-160 750
751
Oppedal F Taranger G L and Hansen T (2003) Growth performance and sexual 752
maturation in diploid and triploid Atlantic salmon (Salmo salar L) in seawater tanks exposed 753
to continuous light or simulated natural photoperiod Aquaculture 215 145-162 754
755
Pelster B (1999) Environmental influences on the development of the cardiac system in fish 756
and amphibians Comp Biochem Physiol Part A Mol Integr Physiol 124 407-412 757
758
Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759
Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760
integrated optodes Physiol Biochem Zool 86(5) 588-592 761
762
Ramaglia V and Buck L T (2004) Time-dependent expression of heat shock proteins 70 763
and 90 in tissues of the anoxic western painted turtle J Exp Biol 207 3775-3784 764
765
Randall D J (1982) The control of respiration and circulation in fish during exercise and 766
hypoxia J Exp Biol 100 275-288 767
768
Richards J G (2009) Metabolic and Molecular responses of fish to hypoxia In Fish 769
Physiology Vol 27 (ed J G Richards A Farrell and C Brauner) pp 443-486 Elsevier - 770
Academic Press Amsterdam 771
772
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
27
Rombough PJ (1988a) Growth aerobic metabolism and dissolved oxygen requirements of 773
embryos and alevins of steelhead Salmo gairdneri Can J Zool 66 651-660 774
775
Sacobie CFD Glebe BD Barbeau MA Lall SP and Benfey TJ (2012) Effect of 776
strain and ploidy on growth performance of Atlantic salmon Salmo salar following seawater 777
transfer Aquaculture 334-337 58-64 778
779
Sadler J Pankhurst P M and King H R (2001) High prevalence of skeletal deformity 780
and reduced gill surface area in triploid Atlantic salmon (Salmo salar L) Aquaculture 198 781
369-386 782
783
Salo D C Donovan C M and Davies K J A (1991) HSP70 and other possible heat-784
shock or oxidative stress proteins are induced in skeletal-muscle heart and liver during 785
exercise Free Radic Biol Med 11 239-246 786
787
Schlesinger M J (1990) Heat-shock proteins J Biol Chem 265 12111-12114 788
789
Schultz R J (1980) Role of polyploidy in the evolution of fishes In Biological Relevance 790
Polyploidy (ed W H Lewis) pp 313-340 New York Plenum Press 791
792
Shao Jun D Gong Z Fletcher G L Shears M A King M J Idler D R and 793
Hew C L (1992) Growth enhancement in transgenic Atlantic salmon by the use of an all 794
fish chimeric growth hormone gene construct Biotechnology 10 176-181 795
796
Smolka M B Zoppi C C Alves A A Silveira L R Marangoni S Pereira-Da-797
Silva L Novello J C and Macedo D V (2000) HSP72 as a complementary protection 798
against oxidative stress induced by exercise in the soleus muscle of rats Am J Physiol 799
Regul Integr Comp Physiol 279 R1539-R1545 800
801
Stevens E D and Sutterlin A (1999) Gill morphometry in growth hormone transgenic 802
Atlantic salmon Environ Biol Fishes 54 405-411 803
804
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
28
Stevens E D Sutterlin A and Cook T (1998) Respiratory metabolism and swimming 805
performance in growth hormone transgenic Atlantic salmon Can J Fish Aquat Sci 55 806
2028-2035 807
808
Stillwell E J and Benfey T J (1996) Hemoglobin level metabolic rate opercular 809
abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis) 810
Fish Physiol Biochem 15 377-383 811
812
Stokstad E (2002) Transgenic species Engineered fish Friend or foe of the environment 813
Science 297 1797-1799 814
815
Taylor J F Preston A C Guy D and Migaud H (2011) Ploidy effects on hatchery 816
survival deformities and performance in Atlantic salmon (Salmo salar) Aquaculture 315 817
61-68 818
819
Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820
Fish Biol Fish 14 391-402 821
822
Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823
temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824
cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825
826
Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827
Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828
829
Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830
Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831
2737-2744 832
833
Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834
hatchery stress in salmon Aquaculture 223 175-187 835
836
837
838
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
29
Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839
salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840
transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841
Different letters between groups indicate significant differences at P lt 005 while their 842
absence indicate no statistical difference 843
844
2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)
total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002
yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001
yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002
caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16
total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08
845
846
Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847
combination with the optophysiological system to simultaneously measure metabolic rate 848
(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849
alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850
was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851
time M O2 was calculated from the time integral of the gas concentration curve derived from 852
the PO2 recording after flushing the chamber 853
854
Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855
Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856
transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857
8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858
determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859
normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860
differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861
Data shown are means plusmn SE for the PO2 ranges specified 862
863
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
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EPTE
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MA
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IPT
30
Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864
diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865
in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866
panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867
5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868
significant difference (P lt 005) in respective parameters compared to normoxic (control) 869
conditions All values are expressed as mean plusmn SE 870
871
Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872
triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873
(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874
group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875
graphs depict protein band density obtained from densitometric scans of immunoblots The 876
asterisks indicate a significant difference (P lt 005) from respective control conditions within 877
each genotype All values are expressed as mean plusmn SE 878
879
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
enta
l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
nal o
f Exp
erim
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l Bio
logy
ndash A
CC
EPTE
D A
UTH
OR
MA
NU
SCR
IPT
The
Jour
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ndash A
CC
EPTE
D A
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NU
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IPT
The
Jour
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ndash A
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MA
NU
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IPT
The
Jour
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NU
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IPT
The
Jour
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ndash A
CC
EPTE
D A
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MA
NU
SCR
IPT
The
Jour
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ndash A
CC
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D A
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OR
MA
NU
SCR
IPT
The
Jour
nal o
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l Bio
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ndash A
CC
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D A
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OR
MA
NU
SCR
IPT
The
Jour
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ndash A
CC
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D A
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IPT
The
Jour
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ndash A
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The
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