J Exp Biol Advance Online Articles. First posted online on ... · 17 behaviour of the red swamp...
Transcript of J Exp Biol Advance Online Articles. First posted online on ... · 17 behaviour of the red swamp...
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Physiological and agonistic behavioural response of Procambarus clarkii to an 1
acoustic stimulus 2
3
Monica Celi1, Francesco Filiciotto2, Daniela Parrinello1, Giuseppa Buscaino2,*, Alessandra 4
Damiano1, Angela Cuttitta2, Stefania D’Angelo3, Salvatore Mazzola2, Mirella Vazzana1 5
6 1Laboratory of Marine Immunobiology, Department of Environmental Biology and Biodiversity, Division of Animal 7
Biology and Anthropology, University of Palermo, Via Archirafi 18, Palermo, Italy 8 2Istituto per l’Ambiente Marino Costiero UOS Capo Granitola — National Research Council, Via del Faro No. 3 - 9
TG, 91021, Campobello di Mazara (TP), Italy 10 3 WWF Italia, via Lo Zano n. 29, 91026 Mazara del Vallo (TP), Italy 11
12
*To whom correspondence should be addressed: [email protected] 13
14
Abstract 15
This study examined the effects of an acoustic stimulus on the haemolymph and agonistic 16
behaviour of the red swamp crayfish Procambarus clarkii. The experiment was conducted in a 17
tank equipped with a video recording system using 6 groups (3 control and 3 test groups) of five 18
adult crayfish (30 specimens in total). After one hour of habituation, the behaviour of the crayfish 19
was monitored for two hours. During the second hour, the animals in the test groups were exposed 20
to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dBrms re 1 µPa at 12 kHz) 21
acoustic stimulus for 30 minutes. Exposure to the noise produced significant variations in 22
haemato-immunological parameters as well as a reduction in agonistic behaviour. 23
24
INTRODUCTION 25
More than 500 recognised species of crayfish are distributed in aquatic habitats of all substrata 26
types across all continents except Antarctica and Africa (Taylor, 2002). Shelters range from 27
natural assemblages of rocks to constructed burrows in mud or sand. The red swamp crayfish 28
Procambarus clarkii (Girard, 1852) is an invasive freshwater species that originated in the south-29
central United States and currently shows a cosmopolitan distribution. This species has been 30
imported to Italy for farming purposes since 1987. Escaped crayfish have invaded natural habitats 31
and become stabilised in many ponds, lakes, and streams across Italy in recent years (Gherardi et 32
al., 1999). Although this crayfish is an aquatic species, it is highly resistant to air exposure and is 33
able to survive for several days outside the water (McMahon and Stuart, 1999). Several eco-34
ethological features of P. clarkii explain its rapid spread in the wild. The species’ biological cycle 35
reflects the hydrogeological cycle and water temperature changes in the invaded areas (Gutierrez-36
http://jeb.biologists.org/lookup/doi/10.1242/jeb.078865Access the most recent version at J Exp Biol Advance Online Articles. First posted online on 1 November 2012 as doi:10.1242/jeb.078865
Copyright (C) 2012. Published by The Company of Biologists Ltd
http://jeb.biologists.org/lookup/doi/10.1242/jeb.078865Access the most recent version at First posted online on 1 November 2012 as 10.1242/jeb.078865
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Yurrita et al., 1999). This crayfish is also highly resistant to environmental stress, including 37
extreme temperatures (Gherardi and Holdich, 1999; Paglianti and Gherardi, 2004), the absence of 38
water, salinity, low oxygen concentrations, and the presence of pollutants (Gherardi et al., 2002). 39
The success of P. clarkii as an invader is further supported by its generalist feeding habits as well 40
as its competitive superiority over native species due to its larger and big dimensions and high 41
grip claws and highly aggressive behaviour (Gherardi and Cioni, 2004). Individuals of both sexes 42
and assorted sizes usually live together, and social activity is particularly notable from spring to 43
autumn. During this period, adult individuals commonly leave their burrows after sunset and move 44
around the populated area. Crayfish exhibit agonistic behaviour when competing for habitats, 45
shelters, mates, and food (Bergman and Moore, 2003). The primary result of such agonistic 46
interactions is the establishment of a dominance relationship that can alter each individual’s access 47
to resources. Aggressive encounters between individuals (agonistic behaviour) are very common 48
(Bergman and Moore, 2003; Buscaino et al, 2012). The crayfish touch each other and assume 49
stereotyped postures aimed at threatening the opponent (Graham and Herberholz, 2009). Crayfish 50
have been used as a behavioural model system to study aggression (Dingle, 1983; Hyatt, 1983) 51
because of their very efficient (big dimensions and high grip) chelipeds (Garvey and Stein, 1993; 52
Schroeder and Huber, 2001) and the ritualised nature of their agonistic fights (Bruski and 53
Dunham, 1987). In particular, due to the high frequency of the agonistic behaviour in 54
Procambarus clarkii (Bergman and Moore, 2003; Buscaino et al, 2012), the observation of the 55
agonistic event and the motility (as a factor that could further the agonistic encounters) could 56
evidence alteration in the baseline behaviour due to external factor, such as an acoustic stimulus. 57
Moreover, the red swamp crayfish that is characterized by a high resistance to environmental 58
stress, could serve as a good model to examine the impacts of acoustic stimuli on behavioural 59
dynamics and the physiological parameters that reflect stress conditions. In addition, because P. 60
clarkii emits acoustic signals in both air and underwater (Favaro et al., 2011; Buscaino et al., 61
2012), it is possible that in this species sound (pressure variation and/or particle movements) can 62
play an ecological role (e.g. predator or conspecific movements perception) so that make these 63
animals sensible to an acoustic stimulus. Otherwise, some studies have evaluated the effects of 64
very high sound pressure levels stimuli (air guns used for seismic surveys) on marine crustacean 65
behaviour and biochemical parameters such as haemocytes, serum proteins, and enzymes without 66
significant effects (Christian et al., 2003; Andriguetto-Filho et al., 2005). While, Payne et al. 67
(2007) found that lobster exposed to very high as well as low sound level had experienced no 68
effect on delayed mortality or damage to mechanosensory system associated with animal 69
equilibrium and posture. However sub-lethal effects were observed with respect to feeding and 70
serum biochemistry with effect sometimes being observed weeks to months after exposure. 71
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Crustaceans might experience pain and stress in ways that are analogous to those all vertebrates 72
(Elwood et al., 2009). Potentially painful stimuli applied to vertebrates typically produce 73
vegetative modifications (behavioural changes as avoidance reaction), changes in blood flow, 74
respiratory patterns, biochemical and endocrine changes (Elwood et al., 2009). There has, 75
however, been limited examination of similar responses in crustaceans. 76
The behavioural observations in combination with physiological assessment, could give a 77
complete understanding of an external stimulus impact on an organism/population/species. In 78
particular, this combination could assume a significant relevance in crustaceans where the 79
behavioural patterns in response to stress condition are not yet well known. For example, 80
behavioural, physiological and biochemical adaptations have been identified in cave crayfish such 81
as, a decrease in locomotion and oxygen consumption, as well as a decrease in metabolic rates 82
after exposure to environmental stress (Caine, 1978). One possible avenue for evaluation of the 83
impact of an external stimulus is through the cardiac and respiratory systems. It is well known that 84
autonomic control of the respiratory and cardiovascular systems can regulate oxygen availability 85
and nutrients to specific target tissues needed for an impending behavioural response. On this 86
regard, Schapker et al., (2002) showed that crayfish rapidly alter heart rate (HR) and ventilatory 87
rate (VR) with changes in the environment and that HR and VR indicators were far more sensitive 88
than behavioural data alone. Moreover, in crayfish Bierbower (2010) used the tail flip response in 89
combination to HR and VR as bioindexes of the whole animal status to CO2 exposure as 90
environmental stressor. Specifically, the author observed a repellent/avoidance behaviour that 91
could be the result of avoiding the paralytic action resulting with CO2 exposure and a decrease 92
until cessation of HR and VR in correlation with CO2 increasing levels. In fish, Buscaino et al. 93
(2010) showed the relationship between behaviour and haematological parameters in relation to a 94
noise exposure. In particular, this short-term noise experiment showed an increase in motility and 95
glucidic metabolism of sea bream and sea bass. Hyperglycemia is a typical response of many 96
aquatic animals exposed to an external stress stimuli. In particular, in crustaceans increased 97
circulating crustacean Hyperglycemic Hormone (cHH) titres and hyperglycemia are reported to 98
occur following exposure to several environmental stressors (Durand et al., 2000; Lorenzon et al., 99
2002). Moreover, environmental stress seems to be an important factor for determining reduction 100
of immunocompetence with increasing prevalence of disease in crustaceans (Sinderman, 1979). 101
Several immune mechanism in Crustacea have been described and they derived essentially from 102
haemolymph cells (Destomieux et al., 1997). 103
Haemolymph cells play a central role in the immune mechanisms in Crustacea (Soderhall 104
and Smith, 1983; Hose and Martin, 1989; Hose et al., 1992; Smith and Chisholm, 1992; Clare and 105
Lumb, 1994; Destoumieux et al., 1997). Three cell types, hyaline, semigranular and granular cells, 106
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are commonly recognised in crustaceans (Bauchau, 1981; Tsing et al., 1989; Hose et al., 1990) 107
and are involved in coagulation, phagocytosis and the production of melanin by the 108
prophenoloxidase (proPO) system. Haemocytes are activated by microorganisms (Vargas-109
Albores, 1995; Vargas-Albores, et al. 1997) and are involved to eliminate foreign particles (Hose 110
and Martin, 1989; Bachère et al., 1995). The immune responses include the release of 111
peroxinectin, from the blood cells. This protein is involved in cell adhesion, degranulation, 112
opsonic, and peroxidase activity (Johansson et al., 1995). Others proteins, involved in defence 113
mechanisms are the stress proteins, also known as heat shock proteins (Hsps), are a highly 114
conserved class of proteins that show elevated expression during periods of stress in organisms as 115
phylogenetically divergent as bacteria and humans. Hsp70 is present at low levels in many cells 116
but is highly induced by stress, regardless of the stage of the cell cycle (Hang and Fox, 1996). In 117
decapod crustacean larvae, the elevation in Hsp70 expression was prolonged depending on the day 118
of pesticide exposure. This effect was directly related to the observed increase in mortality 119
(Snyder and Mulder, 2001). Liberge and Barthélémy (2007) showed that heat stress induced the 120
expression of Hsp70 and superoxide dismutase in the shell glands (structures involved in 121
reproduction) and, more particularly, during the formation of the diapause egg envelope in 122
Hemidiaptomus roubaui (Copepoda, Crustacea). The modulation of certain immunological and/or 123
physiological parameters in response to stressful conditions may serve as an important indicator of 124
health status (Perazzolo et al., 2002). 125
In this context, our understanding of crustacean immune mechanisms and the signals that 126
trigger haemolymph cells (Jiravanichpaisal et al., 2006) together with behavioural observation 127
could provide to monitor the effects of stress factors. In this study, we measured changes in 128
agonistic behaviour and haemolymph parameters in red swamp crayfish (Procambarus clarkii) 129
exposed to 30 minutes of an acoustic stimulus using the motility, the n. of tail flip, the n. of fights 130
and the analyses of total and differential haemocyte counts (THC and DHC, respectively), 131
glycaemic serum levels, total serum protein concentration (PC) and Hsp70 protein expression 132
levels. 133
134
MATERIALS AND METHODS 135
Collection and housing of animals 136
Thirty adult red swamp crayfish (Procambarus clarkii) (17 males and 13 females) weighing 26.1 137
± 9.3 g (mean ± SD) and measuring 9.4 ± 1.0 cm in total length and 4.7 ± 0.6 cm in carapace 138
length were used for this study. The crayfish were captured at the Preola and Gorghi Tondi 139
Natural Reserve (NW Sicily) and acclimated for one month at the Capo Granitola/CNR laboratory 140
(SW Sicily) in 2 shaded, outdoor PVC circular tanks (3.0 m in diameter, 1.0 m in depth) supplied 141
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with a thin layer of sand (1 cm deep). The temperature and salinity levels were monitored using a 142
multiparametric probe (556 MPS, YSI incorporated, USA) and kept constant at 24.02 ± 0.38°C 143
(mean ± SD) and 0.9 ± 0.01 ppt (mean ± SD), respectively, with a constant flow of water at a rate 144
of 25 ± 3.7 l min–1 (mean ± SD). The animals were fed pellets and frozen fish ad libitum. The P. 145
clarkii specimens were deprived of food for 2 days before the start of the experimental trials. All 146
animals were kept under natural photoperiods. 147
148
Rationale and experimental procedures 149
The crayfish were randomly collected from the holding tanks in groups of 5 individuals, assigned 150
to the control or test group and used in one experiment only. In total, 6 experimental trials (3 151
controls and 3 tests) were performed in two experimental tanks (control and test tanks) that lacked 152
shelter. 153
The animals, 5 control specimens and 5 test specimens, were simultaneously released into 154
the control and test tanks, respectively (Fig. 1). After a 1-h habituation period, we monitored and 155
video-recording the behaviour of the crayfish for two hours (1h = pre-experimental phase; 30 156
min= during-experimental phase; 30 min= post-experimental phase). In the during-experimental 157
phase individuals in the test groups were exposed to an acoustic stimulus for 30 min. Members of 158
the control group did not receive any stimuli. At the end of the post-experimental phase, both 159
control and stimulated animals were captured with a net and placed on crushed ice for 30 min to 160
induce torpor or “cold anaesthesia” to make sampling of the haemolymph. The samples were 161
immediately collected from five control and five experimental animals, and the crayfish then were 162
transferred into a small tank and released after recovery. This experimental procedure was 163
repeated three times. 164
165
Acoustic stimulus 166
Although the ability of the red swamp crayfish to perceive acoustic signals is unknown, this 167
species is able to generate wide-band pulse in air (Favaro et al. 2011) and in water (Buscaino et 168
al., 2012). Based on the idea that animals that produce acoustic signals may be able to perceive 169
said signals (e.g., for conspecific movements perception or communication), we decided to use a 170
stimulus with frequencies contained in both of the signals (air and aquatic environment) produced 171
by P. clarkii. The acoustic stimulus was therefore set to emit at a frequency band of 0.1-25 kHz. 172
Moreover, in the natural environmental, this band frequency is mainly produced by vessel traffic 173
(Sarà et al., 2007). 174
A 10-second linear sweep with a peak amplitude of 148 dB re 1 µPa rms at 12 kHz was 175
used to cover the selected frequency band (see Fig. 2). The linear sweep was repeated for 30 min 176
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without pause. The signals were generated by a waveform generator (Model 33220A, Agilent 177
Technologies, Santa Clara, CA, United States) connected to an underwater moving coil 178
loudspeaker (Model UW30, Lubell, Columbus, Ohio, USA) with a 100 Hz – 10 kHz-rated 179
frequency response. 180
The acoustic stimulus was recorded using a calibrated hydrophone (model 8104, Bruel & 181
Kjer, Nærum, Denmark) with a sensitivity of -205.6 dB re 1V/μPa ± 4.0 dB in the 0.1 Hz – 80 182
kHz frequency band. The hydrophone was connected to a digital acquisition card (USGH416HB, 183
Avisoft Bioacoustics, Berlin, Germany; set with a 40-dB gain) managed by dedicated Avisoft 184
Recorder USGH software (Avisoft Bioacoustics, Berlin, Germany). 185
The signals were acquired at 300 kilosamples per second at 16 bits and analysed by the 186
Avisoft-SASLab Pro software (Avisoft Bioacoustics, Berlin, Germany). The digital acquisition 187
card was calibrated with pure tone sine waves at different frequencies (1 and 20 kHz) and 188
different intensities (peak-to-peak 0.1 and 0.5 V) produced by a signal generator (AGILENT 189
33220, United States) using the SASLab Pro software. 190
191
Video monitoring system and analysis 192
To avoid disturbing the animals, we placed the equipment required for video monitoring and 193
recording in a laboratory located 5 m away from the tank. The video monitoring was carried out 194
using a low light cameras (Model CCD colour camera 1090/205, Urmet Domus SPA) placed 195
above the centre of the tanks for an overall view of the experimental space (Fig. 1). The signals 196
from the cameras were digitised and stored using a DAQ card (Model DV-RT4 Real Time, D-197
Vision) managed by custom-written software (Model DSE, D-Vision). 198
The video data were analysed in continuous mode. We identified the agonistic behavioural 199
events reported in other decapods (Buscaino et al., 2011a) and other Procambarus species 200
(Bergman and Moore, 2003; Buscaino et al., 2012): fight and tail flip. Moreover, we considered 201
“encounter” when a specimen approached another one without any threat display (Bergman and 202
Moore, 2003; Buscaino et al., 2012). 203
The fight was considered the approach between two or more specimens that continued in 204
series of agonistic activities including: a) the contact with chelae and progressing to pushing with 205
closed chelae, b) opened chelae used to grab an opponent until c) the most intense interaction in 206
which an individual appears to attempt to injure or injiure an opponent by grasping at chelae, legs, 207
or antennae (Bergman and Moore, 2003). The achievement of one or more of these behavioural 208
stages in continuous progression was considered as a single fight event. 209
The tail flip is a typical avoidance-behaviour event consisting in a rapid abdominal flexion 210
resulting in a new position away from the opponent and in crustaceans is highly associated with 211
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sound production (Buscaino et al., 2011a, b; Buscaino et al., 2012). 212
The total number of events (encounter, fight and tail flip) was counted each 6 minutes. 213
Moreover, because the number of encounters/fights could be influenced by the motility, at 214
the end of 6 minutes interval the number of specimen in movements (walking) or stopped (resting) 215
was counted. 216
The observers that analysed the video did not know if them were referred to the pre, during 217
or post experimental phase, neither control or acoustic treatment. 218
These events are behavioural indices that are useful for assessing intraspecific interactions 219
in decapods (Bergman and Moore, 2003). Variations in these events represent alterations of the 220
baseline activities. 221
222
Haemolymph analysis 223
Haemolymph sampling 224
One millilitre of haemolymph was drawn from the ventral sinus between the first and second 225
abdominal segments using a 2-ml syringe fitted with a 23-gauge needle. To delay or prevent 226
coagulation, the syringe was filled with an equal volume of anticoagulant. After the cellular 227
counts the samples were centrifuged at 800 g for 10 min at 4°C to obtain plasma serum and pellets 228
which were stored at -20°C for further use. 229
230
Total (THC) and differential (DHC) haemocyte counts 231
The total number of haemocytes per mm3 (THC) was determined using a Neubauer 232
haemocytometer chamber. Haemocytes were classified according to Lanz et al. (1993) using the 233
presence or absence of cytoplasmic granules as simple criteria. To perform the differential 234
haemocyte count (DHC, %), a small drop of haemolymph was smeared on a slide, fixed in 235
absolute methanol for 6 min, stained with diluted May-Grünwald-Giemsa (3 min in 10-fold 236
diluted May-Grünwald and 10 min in 10-fold diluted Giemsa), dehydrated with absolute ethanol 237
(1 min) and xilene (6 min) and then mounted in permount. Cells were counted in random areas on 238
each slide, and the relative proportions of various classes were computed (Mahmood and Yousaf, 239
1985). A total of 200 cells were counted on each slide. DHCs were calculated using the following 240
equation: 241
DHC (%) = number of different haemocyte cell typestotal haemocyte cells counted
x 100DHC (%) = number of different haemocyte cell typestotal haemocyte cells counted
x 100 242
243
Scanning electron microscopy (SEM) 244
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Haemolymph samples mixed with anticoagulant were dropped directly onto a coverslip pretreated 245
with 0.1% poly-l-lysine. The adherent monolayer was fixed in cacodylate buffer (0.1 M, pH 7.3) 246
containing 2.5% glutaraldehyde, post-fixed in osmium tetroxide (1%), dehydrated in a graded 247
alcohol series and dried at the critical point. The samples were mounted on stubs, gold-coated in a 248
sputter coater and observed by SEM (LEO 420). 249
250
Glucose, osmolarity, and protein assessment 251
Glucose levels were measured with the Accutrend GC kit (Boehringer Mannheim, Germany). 252
Osmolarity was estimated with an osmometer (Roebling, MESSTECHNIK, Berlin, Germany). 253
The total protein concentration of the crayfish haemolymph serum plasma was estimated using the 254
Bradford method (1976). Bovine serum albumin was used like protein standard. 255
256
Haemagglutination assay 257
The haemagglutinating activity (HA) of two-fold diluted samples was assayed in a 96-well 258
microtitre U plate containing a 1% rabbit red blood cells (RRBC) or sheep red blood cells (SRBC) 259
suspension in PBS (PBS-E: 6 mM KH2PO4, 0.11 mM Na2HPO4, 30 mM NaCl, pH 7.4). 260
Erythrocytes were supplied by the Istituto Zooprofilattico della Sicilia (Palermo, Italy) and 261
maintained in sterile Alsever’s solution (27 mM sodium citrate, 115 mM D-glucose, 18 mM 262
EDTA, and 336 mM NaCl in distilled water, pH 7.2). Tris-buffered saline (TBS; see below) 263
enriched with 1% RRBC and SRBC with 0.1% (w/v) gelatin was used as the reaction medium. 264
Twenty-five microlitres of plasma were mixed with an equal volume of RRBC or SRBC 265
suspension and incubated at 37°C for 1 h. Divalent cation requirements were estimated by adding 266
CaCl2 or MgCl2 to the reaction medium, up to a final concentration of 5-10 mM. The titre of the 267
haemagglutinating activity (HT) was expressed as the reciprocal of the highest dilution showing a 268
positive score for agglutination. 269
TBS was used in place of plasma for the negative controls. Each assay was performed in 270
duplicate using serum samples from different specimen preparations. The HA titre was expressed 271
as the average of the recorded values. 272
273
Haemocyte homogenate supernatant preparation (THS) 274
Cells were crushed on ice for 1 h in 1 ml of lysis buffer (RIPA: 0.5% sodium deoxycholate 275
(minimum 97%); 1% NP40; 0.1% SDS with PBS-T (1 M Na2HPO4, 1 M NaH2PO4, 1.5 M NaCl, 276
and 0.1% Tween-20, pH 7.5, supplemented with a cocktail of protease inhibitors: 2 µg/µl antipain, 277
leupeptin and bestatin, 1 µg/µl aprotinin and pepstatin, 1 mM benzamidine, and 0.1 mM AEBSF). 278
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The samples were then centrifuged at 15000 g for 30 min at 4°C. The supernatants were 279
collected and dialysed against 50 mM Trizma base (Tris [hydroxymethyl] aminomethane), pH 7.5, 280
and the protein contents were estimated. 281
282
283
SDS-PAGE and Western blot 284
The equivalent of 25 µg of total lysates for each sample was separated on 7.5% SDS-PAGE under 285
reducing conditions according to the Laemmli method (1970). SDS-polyacrylamide minigels were 286
transferred to nitrocellulose membranes using a semidry transfer apparatus (BioRad) and blocked 287
with 5% bovine serum albumin (BSA) in TBS-T (20 mM Trizma base, pH 7.5, 300 mM NaCl, 288
0.1% (v/v) Tween-20 with 0.02% sodium azide) for 1 h at room temperature (r.t.). According to 289
Celi et al. (2012), the membrane was incubated over night at 4°C with the primary antibody 290
(monoclonal anti-heat shock protein 70 antibody produced in mouse, Sigma Aldrich; 1:800 291
dilution), washed with TBS-T (three times for 5 min each), and incubated with alkaline 292
phosphatase-conjugated goat anti-mouse IgG (1:7500 for 1 h at r.t.). After washing with TBS-T 293
(three times for 5 min each), the membranes were incubated with the 5-bromo-4-chloro-3-indolyl 294
phosphate/nitro blue tetrazolium liquid substrate system (BCIP/NBT). The Alpha Imager software 295
was used for densitometric analysis of the immunoblotted bands. 296
Five specimens from each experimental group (control and test groups, 30 samples in 297
total) were examined, and each test was repeated in triplicate. 298
299
Statistical analysis 300
Because the behavioural data were not normally distributed, the Mann-Whitney U test was used to 301
compare the “encounters”, “fight” and “tail flip” events between control and test groups as well as 302
among pre-, during- and post-experimental phases. An unpaired t-test was used to determine 303
significant differences in plasma glucose, total protein, THC, DHC and Hsp70 expression levels. 304
305
RESULTS 306
Behavioural events 307
A total of 836 behavioural events were recorded during the 6 experimental trials, of which 379 308
were encounters, 380 were fights and 77 were tail flips. No significant differences in behavioural 309
responses were observed between control and test groups in the pre- or post-experimental phases 310
(Fig. 3). Conversely, in during-experimental phase, significant differences in the numbers of 311
encounters, fights and tail flip events were observed between the control and test groups (P < 312
0.05; Fig. 3). In particular, during the acoustic stimulus, the test crayfish exhibited a lower number 313
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of encounters, fights and tail flips as compared with the control animals. No significant 314
differences were found in the motility in during-experimental phase between control and test 315
group, as well as in the ratio of the motility during/pre/post experimental phase for the control and 316
test groups (P > 0.05). 317
318
319
Microscopic observations of circulating haemocytes 320
The haemocyte monolayers were comprised of flattened and well-spread cells and contained three 321
morphologically distinct cell types that could be differentiated by the presence and size of 322
granules. Three types of circulating haemocytes were identified by light and SEM. Granulocytes 323
are ovoid or fusiform and contain large acidophilic granules (Fig. 4a) that give a rough texture to 324
the cell surface (Fig. 4b). Semigranulocytes can be fusiform and generally display a smooth shape 325
(Fig. 4d). These cells contain small cytoplasmic granules that are typically eosinophilic (Fig. 4c). 326
Hyaline cells appear ovoid or fusiform in shape and are characterised by the absence of 327
cytoplasmic granules (Fig. 4e) and a smooth surface (Fig. 4f). 328
329
THC and DHC 330
The number of circulating haemocytes (total haemocyte count, THC) was approximately 4.7x106 331
± 0.4x105 cells ml-1. Hyalinocytes (H) represented approximately 20 ± 2.4% of the total 332
circulating haemocytes, whereas semigranulocytes accounted for 22.5 ± 3.3%, and granulocytes 333
for 57.5 ± 2.2%. 334
Acoustic stimuli significantly affected both THC and DHC. Following the 30-min acoustic 335
stimulus, the THC of the stressed crayfish decreased by approximately 50% relative to the initial 336
count (P < 0.001) (Table1). A different pattern was observed for the DHC. In tested crayfish, a 337
significant increase in hyaline cell number (from 20% to 58%, P < 0.001) was accompanied by 338
significant decreases in the relative proportions of granular and semigranular cells (P < 0.001 and 339
P < 0.01, respectively) relative to the values determined for the control group (Table 1). 340
341
Serological parameters 342
Glucose levels were significantly higher (575 ± 34 mg/dl, P < 0.01) in the test group than the 343
control group (Table 2). However, no differences in osmolarity or total protein content were 344
observed between groups (Table 2). 345
346
Haemagglutination titre 347
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The serum samples from the control crayfish agglutinated both RRBC and SRBC. The highest 348
titre was found with sheep SRBC (7.2 ± 1.66), whereas the RRBC yielded a score of 6.3 ± 2.1. 349
The HA of the serum from stressed specimens was decreased by approximately 50% (P < 0.01) 350
(Table 2). 351
352
Hsp70 protein expression after treatment 353
As shown in Fig. 5A, the anti-mouse Hsp70 mAb cross-reacted with a 70-kDa band in circulating 354
haemocytes from both the control and test groups. A densitometric analysis of Hsp70 protein 355
levels (Fig. 5B) revealed a significant increase in expression in the haemocytes collected from 356
stressed animals. Hsp70 expression peaked (3-fold higher than the untreated samples) after the 357
period of acoustic stimuli. 358
359
360
DISCUSSION 361
This study showed that an acoustic stimulus can reduce the agonistic behaviour of the crayfish 362
Procambarus clarkii, as demonstrated by the significantly reduced numbers of both fights and tail 363
flip events. According to Bergman & Moore (2003), P. clarkii crayfish engage in agonistic 364
interactions with high frequency to establish dominance relationships that regulate access to 365
resources. Similarly, Capelli and Hamilton (1984) have shown that in a laboratory environment, 366
food and shelter affect the agonistic behaviour of the crayfish Orconectes rusticus. In particular, 367
aggressive activity decreases with the increased availability of both shelter and food. To avoid 368
these effects, we observed crayfish held in tanks without shelter and deprived of food for two days 369
before the experimental trials. 370
In the during-experimental phase, although the motility of specimens of test group was lower, we 371
did not observe a significant reduction in comparison to the specimens of the control group. 372
Otherwise, a significant lower number of encounters of test group were observed in the during-373
experimental phase. Similarly to this result, the acoustic stimulus induced a decrease in the natural 374
aggressive activity also (number of fights and tail flip events) of the crayfish. Accordingly, when 375
the acoustic stimulus was interrupted (post-experimental phase), an increase in aggressive 376
agonistic behaviour was observed. 377
Our results indicate that P. clarkii could perceive all or part of the acoustic stimuli used in 378
this study (0.1-25 kHz bandwidth) within the wider bandwidth of their underwater acoustic 379
emissions (Buscaino et al., 2012). However, no data on the anatomical-functional structures with 380
which they detect acoustic energy (such as variation in pressure) are currently available. The 381
sensitivity of aquatic decapods to particle displacement and hydrodynamic stimulation is poor 382
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compared to other aquatic organism like fishes (Breithaupt and Tautz, 1990; Goodall et al., 1990; 383
Popper et al., 2001). From our study is not possible to discern the quantitative and qualitative 384
receptors involved to the perception of the variation of pressure or particle water movement or if 385
the sound produced physical effects on entire animal body without stimulating a specific receptor. 386
Lobster and crayfish primarily respond to hydrodynamic stimulation (behavioural study) than 387
pressure (Goodall et al., 1990; Popper et al., 2001). However, these studies focused on acoustic 388
frequencies lower (20-180 Hz) than our study (0.1-25 kHz). 389
In crustaceans, mechanoreceptors are located in cuticular extensions of the exoskeleton 390
and are called sensilla (Ali, 1987). Decapod mechanoreceptors include setae (hair-like cells), 391
chordotonal organs, and internal statocysts (Popper et al., 2001). In macruran decapods (crayfish), 392
the hairs are sensitive to vibration (Breithaupt and Tautz, 1990) and can respond to frequencies up 393
to 100 Hz. In P. clarkii, the antennules (lateral plus medial flagella) possess both chemosensory 394
setae and mechanosensory setae. The latter respond to hydrodynamic stimuli up to 100 Hz 395
(Breithaupt and Tautz, 1990). In particular, the medial flagellum functions as a hydrodynamic 396
receptor (Horner et al., 2008; Monteclaro et al., 2010). In our study, the animals didn’t show any 397
preference in the choice of the tank’s side to take up (near or far from the underwater loudspeaker) 398
in the during-experimental phase. Otherwise, is possible that the reverberation/reflection effects of 399
the sound inside the tank make animals unable to detect the sound source, inhibiting them to 400
perform the avoidance behaviour. Moreover, the agonistic behaviours were reduced during the 401
stimulation probably in consequence of the impact of an external stress condition that may inhibit 402
aggressive state as a result of a preservation instinct. A suggestion could be that crayfishes give 403
the priority to the external stimuli (considered as a stress source and confirmed by the increase of 404
glycaemic and Hsp70 levels and decrease of THC) respect other baseline agonistic behaviours. 405
In marine shrimps and crabs (Christian et al., 2003; Andriguetto-Filho et al., 2005), 406
exposure to stronger acoustic stimuli (air guns) produced no obvious effects on behaviour or 407
biochemical parameters (serum proteins, serum enzymes, calcium, and haemocyte types). 408
Otherwise, Payne et al. (2007) observed sub-lethal effect as well as feeding rate on lobsters 409
exposed to air gun. The discrepant findings of these prior studies and the present investigation 410
suggest that the effects of acoustic stimuli are perceived differently under different environmental 411
conditions (e.g. tank or natural environment, distance from the acoustic source) and acoustic 412
typologies (e.g. source level, frequency, duration) or even between different crustacean species. In 413
fish, Santulli et al. (1999) reported that exposure to air gun blasts affected biochemical parameters 414
(cortisol, glucose, lactate, AMP, ADP, ATP, and cAMP) in sea bass, and Buscaino et al. (2010) 415
showed that the exposure of sea bass and gilthead sea bream to a 0.1-1 kHz linear sweep (150 416
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dBrms re 1 µPa) caused a significant increase in motility that influenced haematological 417
parameters. 418
In aquatic crustaceans, various types of stress, including hypoxia (Le Moullac et al., 1997, 419
1998), low salinity (Perazzolo et al., 2002), viral infection and administration of an 420
immunostimulant (Hennig et al., 1998; Sritunyalucksana et al., 1999), affect haematological 421
parameters. In aquatic animals, hyperglycaemia is a typical stress response to harmful physical 422
and chemical environmental changes, including hypoxia and exposure to air during commercial 423
transport (Spicer et al., 1990; Zou et al., 1996; Kuo and Yang, 1999; Morris and Oliver 1999; 424
Durand et al., 2000; Speed et al., 2001). Hyperglycaemia has been associated with increased 425
circulating crustacean hyperglycaemic hormone (CHH) titres (Lorenzon et al., 1997, 2002; 426
Durand et al., 2000; Santos et al., 2001) and has been used as an index to assess CHH activity and 427
environmental stress (Webster, 1996; Bergmann et al., 2001; Toullec et al., 2002). Accordingly, in 428
P. clarkii, acoustic stress led to a significant increase (P < 0.01) in haemolymph glucose levels. 429
However, exposure of P. clarkii to an acoustic stimulus no significant effects on internal 430
osmoregulatory capacity and on total serum protein concentration (PC). In aquatic crustaceans and 431
particularly in decapods, the organs of the branchial chambers are the primary source of the 432
osmotic and ionic regulation (Péqueux, 1995). Exposure to environmental stressors and 433
pathological agents on osmoregulation usually, in crutacean, results in a decrease of its Na+ and 434
Cl- regulation. The partial or complete loss of osmoregulatory and ionoregulatory capacity is 435
generally linked to distruptions of the osmotic and ionic regulations. Studies on crustacean 436
responses to various environmental stressors revealed that the effect of stress upon osmotic and 437
ionic metabolism was time and dose-dependent (Charmantier et al., 1989; Charmantier and Soyez, 438
1994; Lignot et al., 2000). 439
Changes in the protein composition of haemolymph has been used like a stress indicator to 440
monitor shrimp health status and exposure to environmental stress (Chen et al., 1994; Chen and 441
Cheng, 1995) seem to depend from certain physiological and environmental variables (Bursey and 442
Lane, 1971; Chen and Cheng, 1993; Chen et al., 1994; Chen and Cheng, 1995), sex and animal 443
size (Chen and Cheng, 1993). 444
Naturally occurring agglutinins, including those with erythrocyte targets 445
(haemagglutinins), are involved in innate immunity in invertebrates (reviewed in Marques and 446
Barracco, 2000). In our study, acoustic stress induced a significant decrease in the agglutinating 447
titre of P. clarkii serum, as assayed with sheep and rabbit erythrocytes. Further analyses using 448
sugar inhibition might elucidate whether the serum haemagglutinins are lectins (Sharon, 2007). 449
In crustaceans, haemocytes are involved in organismal homeostasis and manage several 450
immune functions, including coagulation, phagocytosis, degranulation, opsonisation and 451
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production of melanin by the prophenoloxidase (proPO) system (Vargas-Albores, 1995; Vargas-452
Albores et al., 1997). According to Lanz et al. (1993), P. clarkii granular and semigranular 453
haemocytes may participate in the proPO system as well as phagocytic or cytotoxic functions. 454
THCs and DHCs have been used to assess crustacean health and the effects of stressful 455
conditions (Jussila et al., 1997). Decreases in the THC under stressful conditions have been 456
reported for several marine crustacean species (Smith et al., 1995; Hennig et al., 1998; Le Moullac 457
et al., 1998; Sánchez et al., 2001). Similarly, under acoustic stimuli, the THCs of our P. clarkii 458
specimens were reduced, suggesting the possibility of immune depletion as well as an increased 459
risk of infection. Moreover, acoustic stimuli also altered the DHC (relative proportions of HC, 460
SGC and SGC in the THC). Although the response of the DHC to different stressors is not well 461
understood, it has been used as a stress indicator in crustaceans (Jussila et al., 1997; Johansson et 462
al., 2000). Acoustic stimuli resulted in an increase in the relative number of hyaline cells and 463
decreases in semigranulocytes and granulocytes. These results are similar to those reported in the 464
literature (Jussila et al., 1997; Fotedar et al., 2001, 2006), where the proportions of granulocytes 465
and semigranulocytes were lower in moribund lobsters than healthy individuals (Bauchau, 1981; 466
Sequeira et al., 1995). 467
Stress proteins, also known as heat shock proteins (Hsps), are a highly conserved class of 468
proteins that show elevated transcription during periods of stress in organisms as phylogenetically 469
divergent as bacteria and humans. These proteins have been shown to play numerous important 470
roles in maintaining organismal health, e.g., in the host responses to environmental pollutants and 471
food toxins as well as the development of inflammation. In shrimp, Hsps are involved in the 472
specific and non-specific immune responses to bacterial and viral infections (Roberts et al., 2010). 473
In particular, Hsp70 acts to repair damage to proteins following acute stress and thus plays a key 474
role in cytoprotection (Feder and Hofmann, 1999). 475
In crustaceans, Hsp70 expression serves as a good bioindicator of stressful conditions, 476
including pesticide exposure and heat stress (Snyder and Mulder, 2001; Chang, 2005; Liberge and 477
Barthélémy, 2007). Little is known about the effects of noise on Hsp expression. Wu et al. (2001) 478
showed that Hsp70 expression increased after exposure to a stressful noise in humans, and 479
Hokestra et al. (1998) reported that the expression of Hsp70 (but not Hsp30, Hsp60, or Hsp90) is 480
increased in birds after exposure to a loud noise. We have also recently detected increased Hsp70 481
expression in fish Chromis chromis after exposure to sounds similar to those resulting from 482
human activities (Celi et al., unpublished). In the present study, we show for the first time that 483
acoustic stimuli induce Hsp70 overexpression in P. clarkii haemocytes as expression of a stress 484
status. 485
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In conclusion, exposure to acoustic stimuli altered certain aggressive behavioural patterns 486
and components of the haemato-immunological system of P. clarkii. Among the assessed 487
haemato-immunological parameters, the serum glucose concentration, PC, agglutinating activity, 488
THC, DHC and Hsp70 expression are the most promising parameters reflecting stress status in 489
crayfish. The haemato-immunological responses to the stressful conditions occur in conjunction 490
with behavioural changes. 491
In most natural aquatic environments, the soundscape has been permanently altered due to 492
anthropogenic activities (e.g., traffic vessels, wind turbines, electroacoustic instruments for 493
exploration and navigation), and the impact on aquatic organisms should be investigated over 494
brief, medium and long-term exposure periods (Payne et al., 2007). 495
However, in according with Goodall et al. (1990), further studies should be also performed 496
in a open controlled natural environment (where acoustical field is not influenced by walls such as 497
in the small tanks) to increase the information about the effects of noise on the behavioural and 498
physiological response. 499
500
ACKNOWLEDGEMENTS 501 502 This work was supported by Grants to VM ex 60% from University of Palermo and by the project 503
BIOforIU - PONa3_00025 and RITMARE of IAMC-CNR UO Capo Granitola. 504
505
506
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Liberge, M., Barthélémy, R.M. (2007). Localization of metallothionein, heat shock protein 659
(Hsp70), and superoxide dismutase expression in Hemidiaptomus roubaui (Copepoda, 660
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Perazzolo, L. M., Gargioni, R., Ogliari, P., Barracco, M. A. A. (2002). Evaluation of some 690
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the effect of acclimation. Aquaculture 198, 13– 28. 700
Santos, E. A., Keller, R., Rodriguez, E., Lopez. L. (2001). Effects of serotonin and fluoxetine 701
on blood glucose regulation in two decapod species. Braz. J. Med. Biol. Res. 34, 75- 80. 702
Santulli, A., Modica, A., Messina, C., Ceffa, L., Curatolo, A., Rivas, G., Fabi, G., D’Amelio, 703
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761
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Legends 762
763
764
Fig. 1. Schematic representation of experimental tanks equipped with an underwater loudspeaker 765
and a video camera placed above the centre of the tank. In the control tank, the acoustic stimuli 766
were not emitted by the loudspeaker. 767
768
Fig. 2. Oscillogram (top), spectrogram (middle) and power spectrum (bottom) of the linear sweep 769
emitted 1 m from hydrophone. Oscillogram: pressure (Pascal) vs. time (second). Spectrogram: 770
frequency (kHz) vs. time (s). The intensity is reflected by the gray scale. dB re 1 µParms, 1024-771
sample FlatTop window. Power spectrum: dB re 1 µPa rms (time window 10 sec) vs. frequency (k 772
Hertz). 773
774
Fig. 3. Significant differences in the numbers of behavioural events were only observed between 775
control and test groups during the acoustic stimulus period. Number of fights (A); number of tail 776
flips (B); number of specimens in movement (C); number of encounters (D). Data are expressed 777
as the mean ± SD. Pre = the hour preceding the acoustic stimulus; During = during the acoustic 778
stimulus (30 min) given to the test groups (no acoustic stimulus was administered to the control 779
groups); Post = the period immediately following the stimulus (30 min). 780
781
Fig. 4. Circulating haemocytes from Procambarus clarkii. Light (a, c, e; May-Grϋnwald-Giemsa) 782
and scanning electron microscopy results (b, d, f). (a, b) Granulocytes; (c, d) semigranulocytes; (e, 783
f) hyaline cells. Bars: (a, b) 6 µm; (c, d) 5 µm; (e, f) 3 µm. 784
785
Fig. 5 Effect of the acoustic stimuli on expression levels of the protein Hsp70 in Procambarus 786
clarkii. A: representative western blot of Hsp70 levels in 3 specimens from each group (one for 787
each experimental trial). B: Integrated optical density histogram (IDV) of the Hsp70 protein 788
bands. The data represent the means ± SD (n=15 control and n=15 test specimens). 789
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Table 1. Total haemocyte count (THC) and differential haemocyte count (DHC) in the 1
haemolymph of the control and test groups. The data represent the means ± SD (n=15 control and 2
n=15 test specimens). Significant differences between the control and test groups (acoustic 3
stimulus) are shown (** P < 0.01; *** P <0.001). 4
5 6
7
8
Collection THC Granulocytes
(%)
Semigranulocytes
(%)
Hyaline
(%)
Control Group 4.7x106±0.4x105 57.5±2.2 22.5±3.3 20±2.4
Tests Group 2.2x106±0.5x105*** 30±1.4*** 11.3±0.3** 58±6.2***
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Table 2. Plasma glucose, osmolarity, total protein levels and haemagglutination titre in the 1
haemolymph of Procambarus clarkii exposed to an acoustic stimulus. The data represent the 2
means ± SD (n=15 control and n=15 test specimens). Significant differences between the control 3
and test groups (acoustic stimulus) are shown (** P< 0.01). 4
5
6
7
8
9
10
11
Collection Glucose
(mg/dl)
Osmolarity
(mOsm/kg)
Total protein
(µg/µl)
Haemagglutination titre
RRBC SRBC
Control Group 468±19 400±16 1.18±0.1 6.3±2.1 7.2±1.6
Tests Group 575±34** 395±19 0.95±0.2 3.1±1.0** 3.3±0.9**
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