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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
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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
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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