1/40

Ivar RønnestadDept of Biology, University of Bergen, NORWAY

Gastrointestinal physiology of marine fish larvae

Gastrointestinal physiology of marine fish larvae

• hormonal control• processing capacity• hormonal control• processing capacity

2/40

Topic I:How is the digestive function regulated?(involvement of Cholecystokinin; CCK)

The digestive system

3/40

CCK in fish larvaeAtlantic halibut, bluefin tuna ayu, Atlantic herring

Rotated gut Straight gutHatching

Metamorphosis

mg

fg

hg

posterior

fg hgmg

anterior posterior

anterior

summary

100µm 10µm100µm 10µm

Kam

isak

a.et

al. 2

001,

200

2, 2

003,

200

5.

4/40Kamisaka, Y., Ø. Drivenes, T. Kurokawa, M. Tagawa, I. Rønnestad, M. Tanaka, and J.V. Helvik. 2005. Peptides. 26: 385-393.

Molecular characterisation of CCKTGCGC -61

TTCTTCTCCCTCCTCTTCTCTCCGCTCCGCTTCTCTCTGCTCTCATAGCACGGACTGAAG –1

ATGAACTGTGGCATCTGTGTGTGTGTGCTGCTGGCTGCTCTCTCCAGCAGCGGCTGCCTG 60M N C G I C V C V L L A A L S S S G C L

GGGCTTCCAGCCCACCCCCCGCAGGACGAAGGCCACGCCCGCACAGGGGCTGTCTCCCGG 120G L P A H P P Q D E G H A R T G A V S R

GCAGCGGCGGCAGTACACAGGCGTCACACCCGGTCGGTGGCAGCCCCCAGCGGAGCCCTC 180A A A A V H R R H T R S V A A P S G A L

CTCCCGTTCACCAAAGCAGCCGTGGAGGACGAGGAGGAGCAAGACCCCCGCACCAGCCTT 240L P F T K A A V E D E E E Q D P R T S L

ACCCAGCTCATCGCCACTCTCATCGCAAGGAAAGGTGCGTCCCGCAGGAACTCCGTGCTG 300 T Q L I A T L I A R K G A S R R N S V L

ACCAGCAGAGCCAGTGGGCCGGCCTCCAGCCACAGAATAAAGGACCGGGACTACATGGGC 360 T S R A S G P A S S H R I K D R D Y M G

TGGATGGACTTTGGCCGACGGAGTGCGGAGGAGTACGAGTACTCCTCATAAGACCCACAG 420 W M D F G R R S A E E Y E Y S S *

GCCTGCAGCCTCCCCCATCGTCCAGCACCACCCGTACAGGAGCAGAGATTCCTCATATTT 480

ACTGTTTGGAAAATCATTGTCTGAAAACTATATTTGCGATACATTCGATGTCAATTG 538

603 bp

ORF

137 AA

cDNA

CCK-8

Atlantic herring

5/40

CCK Multiple sequence alignment

Dogfish : HRIADRDYMGWMDFGRRSAEEY-EYAS : 125

Human : HRISDRDYMGWMDFGRRSAEEY-EYPS : 115

Zebrafish1 : HRIKDRDYVGWMDFGRRSAEEY-EYSS : 117

Eel : HRIKDRDYLGWMDFGRRSAEEY-EYSS : 127

Trout-T : HLIKNRDYTGWMDFGRRSAEEN-EHSL : 137

Trout-N : HRIKDRDYNGWMDFGRRSAEEY-EYSL : 138

Puffer1 : HRIADRDYLGWMDFGRRSAEEY-EYSS : 137

Flounder1 : HRIADRDYLGWMDFGRRSAEEY-EYSS : 135

Puffer2 : HRIKDRDYVGWMDFGRRSAEEY-EYSS : 132

Flounder2 : HRIKDRDYLGWMDFGRRSAEEY-EYSS : 135

Herring : HRIKDRDYMGWMDFGRRSAEEY-EYSS : 136

Trout-L : HKIKDRDYLGWMDFGRRSAEEYEEYSS : 132

Catfish : HRIKDRDYLGWMDFGRRSAEEY-EYSS : 123

Goldfish : HRIKDRDYLGWMDFGRRSAEEY-EYSS : 123

Zebrafish2 : HRIKDRDYLGWMDFGRRSAEEY-EYSS : 123

CCK-8

CCK-8

6/40

CC

K c

onte

nt (f

mol

/ in

d)

Days after first feeding

50

0.01

0.1

1

10

0 7 14 21 28

Whole larva

Gut

CCK content in fish larvae

Atlantic halibut

Rojas-García & Rønnestad (2002) J. Fish Biol: 61: 973-986

7/40

CCK content in fish larvae

Standard length (mm)

fmol

CC

K /

ind

(exc

ludi

ng h

ead)

0.0

1.0

2.0

3.0

4.0

5.0

11 12 13 14 15 16

FedFasted

Rojas-García, Morais, Rønnestad (unpubl)

Atlantic herring

8/40

0

20

40

60

80

100

4 8 12 16 20 24 h

Time of hatching (hours)

0

40

80

120

160

200% TCA prec. (incl Protein)

% TCA sol. (incl FAA)cpm/Artemia

% cpm

14C-AA enrichment in Artemia

Morais, Conceição, Dinis and Rønnestad. 2004 Aquaculture. 233: 292-304.

9/40

Protocol

Transfer fed larvae

2 - 24h

Liver

Body (carcass)Body (carcass)

GutGut

Dissection

Incubation water (evacuated)Incubation water (evacuated)

CO2-Trap (metabolised)CO2-Trap (metabolised)

Mor

ais,

Con

ceiç

ão, D

inis

and

Røn

nest

ad. A

quac

ultu

re (

2004

). 23

3: 2

92-3

04.

10/40

AA absorption from prey (Artemia)

0

10

20

30

40

50

60

Gut

Evacuated

0

10

20

30

40

50

60

0 4 8 12 16 20 24

Time after feeding (h)

Body retention

Metabolised

Mor

ais,

Con

ceiç

ão, D

inis

and

Røn

nest

ad. A

quac

ultu

re (

2004

). 23

3: 2

92-3

04.

Atlantic herring

11/40

00,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,91

0 0,1

0,20,3

0,40,5

0,60,70,80,9 1

Tube feeding

The search for a model proteinProduction design:

1 ml moist feed w/ (U)-14C-AA(2mCi / 74MBq)

Tonheim, Espe, Raae, Darias, Rønnestad. 2004. Aquaculture. 235: 553-567

Protein 81 mg/ml3.6 MBq/g

Serum (1,8 ml)

”Salmon Serum Protein”

0,7 mlGelfiltration

+Cut off centrifugation (30 kDa)

Drawing: S Tonheim

12/40

diet with 15 AA 14C labelled Test protein

75 kDa

25 kDa

10 kDa

Highly Hydrolysed Protein

Highly Hydrolysed Protein

Trypsin +endoproteinases

Tonheim, Espe, Raae, Darias, Rønnestad. 2004. Aquaculture. 235: 553-567

Test protein (Intact Protein, Prot)

Test protein (Intact Protein, Prot) Partially Hydrolysed

Protein

Partially Hydrolysed Protein

30 60 120 240

Time forhydrolysis (min)

Pepsin

13/40 Tonh

eim

, Esp

e, H

amre

&R

ønne

stad

. 200

5. J

. Exp

. Mar

. Bio

l.Eco

l. 32

1: 1

9-34

Sum absorbedSum absorbed

0

10

20

30

40

50

60

70

20Hours past tube feeding

0 2 4 6 8 10

% o

f tot

al tr

acer

tube

-fed

Larval experiment

ProtPHPHHP

14/40

Effect of intestinal loading

5 10 15 20 25 30 35 40

Administered protein (μg)

5

10

15

20

25A

bsor

bed

prot

ein

(μg)

R2=0.50

R2=0.98

Tonh

eim

, Esp

e, H

amre

&R

ønne

stad

. 200

5. J

. Exp

. Mar

. Bio

l.Eco

l. 32

1: 1

9-34

ProtPHPHHP

15/40

36 dpff(Larvae)

78 dpff(Juvenile)

Intact protein

Effect of age and stageC

ompa

rtm

enta

l dis

trib

utio

nof

trac

ers

0

20

40

60

80

100

(%)

Atlantic Halibut (12 °C)

Evacuated

Gut

Absorbed

Tonheim, Espe, Hamre & Rønnestad. 2005. J. Exp. Mar. Biol.Ecol. 321: 19-34

Intact protein

Pre-hydrolysedprotein

Larvae ca 25 (dpff)

Tonheim, Espe, Raae, Darias, Rønnestad. 2004. Aquaculture. 235: 553-567

16/40

Absorption rates of FAA

10

100

1000

8 10 12 14 16 18 20 22 24

Standard length (mm)

Gut

Vol

ume

(nl)

Applebaum, Morais, Rojas-García, Rønnestad (unpubl)

Atlantic herring

17/40

Absorption rates of FAA

nmol

FA

A a

bsor

bed

from

0-0

.5 h

r

nmol FAA tube-fed

alaala argarg y = 0.7871x - 0.0889R2 = 0.9923

y = 0.7607x - 0.1218R2 = 0.9783

1

2

3

4

5

6

7

gluglu lyslysy = 0.7787x + 0.0264

R2 = 0.9722

1 2 3 4 5 6 7

y = 0.3499x + 0.1753R2 = 0.6768

1

2

3

4

5

6

7

1 2 3 4 5 6 7

Atlantic Halibut (12 °C)

Applebaum & Rønnestad, Aquaculture. 2004. 230: 313-322

30 min

18/40

Peptide transporters

Pep

4008000

PepNH2

NH2

PepT1; oligopeptidetransporter

Peptidomimmetics

Characterise PepT1Target species: Atlantic cod

Characterise PepT1Target species: Atlantic cod

19/40

Atlantic cod PepT1Characterisation:

• Cloning• Sequencing• Hydropathy analysis• Kinetics (Xenopus expression)

Characterisation:• Cloning• Sequencing• Hydropathy analysis• Kinetics (Xenopus expression)

ORF

755 AA

Rønnestad, Viagas, Gavaia, Cancela (unpubl)

cDNA

20/40

Collaborators:Collaborators:Univ of Algarve, Portugal• Luis EC Conceição• Cláudia Aragão• Maria Teresa Dinis• Leonor Cancela• Paulo J Gavaia• Carla SB Viegas• Sofia Morais*

Kyoto University, Japan• Masaru Tanaka• Masatomo Tagawa• Yuko Kamisaka*

NRIA, Japan• Tadahide Kurokawa• Tohru Suzuki

University of Bergen, Norway• Scott Applebaum• Carlos R Rojas-García*

Institute of Nutrition, Norway• Kristin Hamre• Marit Espe• Sigurd K Tonheim*

IFREMER, France• José L Zambonino-Infante• Chantal Cahu

S. Morais Y. KamisakaC.R Rojas-GarcíaS. K. Tonheim