Huse, Ingvar

Klungsøyr, Leiv

Opstad, Ingegjerd

Walther, Bernt

English

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This paper not to be cited witho~t-Erior reference to the autor. Internatioanl Council for the Exploration of the Sea C .. M.. l 9 8 2 /F : l 7 l'iar i c ul ture Cornmi ttee ENDOCF.INE AND ·NUTRITIONAL FACTORS' A'FFECTING 'THE 'FTRST FOOD UPTAKE BY COD 'LARVAE BY Ingvar Huse and Ing~gjerd Opstad,· Austevoll Marine Aquaculture Stat ion and Leiv Klungsøyr and Bernt Walther, Institute of Biochemistry, University of Bergen. ABSTRACT The gastroendocrinal development of cod laryae was investigated with emphasize oh the rele different hormones from the feed might,play in promoting growth and development or· triggering gastric secretion.. Pepsin like and chymotrypsin like activity was found in unfed larvae, indicating that the digestive system may·besufficen~ly developed. None of the hormones and other substances added to the feed promoted growth. A diet based on cod roe gave significant growth, but no survival :to metamorphosis. - 2 -INTRODUCTION The start feeding of most marine fish is totally dependant on live feed. Even though many larvae readily eat dead food organisms and other inert die~s the larvae will die some time$ The reason for this lack of ability to util dead food not fully under-stood despite the fact that many experiments have been carried out to test different hypotheses concerning this problem. The present study deals with the role different hormones· rnight play in this processe Two hypotheses we.:r;e tested: l. The fish larvae are not fully developed at first feeding, but need a signal subst~nce from the feed to start diegest6n and gro~th. 2. The fish larvae are not fully developed at first feeding, but need a growth-and development promoting substance from the feed. Little is known about substances present in live feed which might influence the developrnent of the digestive apparatus of the fish larvae. Several hormones are known to influe~ce development and differentiation.. vJe picked as possible candid,ates ecdysone and thyroxine, and a synthetic cortisol analog, dexamethasone (D.E. Metzler, 1977) l Production and secretion from ful1y developed digestive enzyme glands is regulated by a number of peptides. The peptide caerulein which is isolated from amphibian skin, has an endocrine activity in teleosts which is similar to the activ~ty of gas€~in and cholecystokinin ln mammal~ (Lei~ Larsson and J.F. Rehfe1d, 1977, G.J .. Dockray, 1979)., J.Fltlchter (1982); demonstrated improved survi val and growth in "whi tef ish ~,~· larvae after addi tion of an acetone extract of quickly frozen· Ar·t·emia· !l·aup·ulii to the feed., -r-1ATERIALS AND METHODS Demons·t-rat.ion ·of ·diges.t·ive ·enzymes· in larva1 extracts. 'P'ep·s·i·n· '1'ike· :a·c:t:ivi·t·y: The fish species studied was cod (Gadus Morhua)., Fr~shly collected larvae were filtered through double layers ~f cheese cloth to remove salt water. The larval mass was frozen and stored without further treatment 0 Extracts were prepared from 6 g o~· larvae in 5 volumes of 100 rnM acetate buffer pH 5., The mixture was hornogenized - 3 and centrifuged at 12 OOO·x g for 15 mine An aliquot of the supernatant was by ul traf il tration ·to a small vol urne, diluted with 10 mM acetate buffer pH 5 and again concentrated. This procedure was twice, and the.whole treatment required 4 days in cold roorn (2 - 4°) The extract was centrifuged and applied to a DEAE (50 x 8 min}equilibrated with 10 rnM aceta_·te buf pH 5 The colum was eluted with 10" 100 200 m.M acetate pH 5., The eluate \vas ana-lyzed fo~ pepsin l activity at pH 2 by Frit.s~h (197~~) with hemoglobin as a modi.fied Lo include a max.imal volume of method of Rick and The rnethod was solution in the incubation mixture, and the incubation was out for 60 min~ Pepsin like act was present in all three fractions of eluate. Enzymefrom a control extract of the stomach of a young cod behaved in a similar way: Same act:ivity was eluted from the DEAE cellulose column when the colurn was washed with 10 mM acetate buffer at pH 5. The bulk of the act~vity appeared in the fractions eluted with 50, 100 and 200 mM acetate buffer (pH 5)~ An extract of ~ g of cod larvae was prepared in 5 volumes of 80 m.M buffer, pH 7e8. The mixture was homogenized and c·entrifuged at 12 000 x g fo'r 15 min.. An aliquot of the extract was treated with 9 pa~ts ·of methano~, and the precipiate removed by centrifugationa The supernatant was tested for chymotrypsin like activity by the method of Rick (1974) with N-benzoyl-L-tyrosine ethyl ester as substr~te., Significant chymotrypsin like activity v1as observed in the methanol treated larval extractQ extract rreshly collected zoopla~cton were frozen in small portions in liquid butane which was again cooled· in liquid nitrogen The lumps of material weie ~eighed and sub-merged in ·4-volurnes of acetoneo The mixture was shaken until all frozen material was thawed .and dispersed The acetone was rernoved in a stream of nitrogen, and the residue was dissolved in a similar volurne og 95% ethanol which was added as such to the feedQ Fertilized eggs were collected a spawning pen and incubated and hatched in polyefhylene nders (Huse Jensen 1980,1981)®. Two to four old la.rvae were transferred to the· 100 l conical experimental tanks with an itial dens t of 20 larvae per l. - 4 -The experimental tanks were supplied with filtered and UV-treated sea water from 55 m depth.. The temperature varied between 6 .. 0°C 'Øiftd .. ~3 .... D°C. during the experimental period.. After each feeding the wa-t.er cfrculation was shut off to enchance feeding stidce{s's.. This led to increased temperature in the.tank water which explains the wide range of temperatures duri~g the experiment. Salinities were relatively stable at 31.2 a· so;do. The different hormones tested were mixed into a standard diet based on hen's egg, proteose pepton, cod liver oil, and fish . ' protein autolysate with v~tamins and minerals added- Particles between 90 and 120j4were made by ~creeni~g with plankton gauze. IIormones were added after this screening. The larvae were fed 2 - 3 times a daya The feed particles stayed in suspension for several hours befare sedimenting. Feed level was kept at more than 2000 particles per l. Each experiment included one group of starving larvae and one group getting only standard diet. Larval samples were collected by tube sampling every second or third day. Feeding incidence, length, myotome height, and dry weight was measured. Five sets of experiments were carried out. The different diets tested are given in table l. TABLE l. Diets and substances tested. EXPERIMENTAL SET NO. No food (starvation gro up·} X X X X X Standard diet (S} .x X X X X S+ Caerulein X S+) :: Dexametha sone X S+ Histamine _X ·s+ Thytoxine X S+ Ecdysone+ Dexamethasone+ Thyroxine+ Caerulein X Squid meal X S+ zooplankton extract X C od roe X Living collected zooplankton X Rotifers - 5 -The substances tested in the standard feed were added in the following concentrations, mg per 500 g of feed: Ecdysone Dexamethasone Thyroxine Caerulein Histamine Zooplankton 100 1000 and 10 100 l 1000 extract 5xl05 -fEthanol was em.porated at room temperature after mixing with standard diet .. RESULTS AND DISCUSSION The development of dry weight over time for larvae fed different diets is given in FIGS l-Se Dry weight is chosen as a growth monitoring parameter as it is more sensitive than length and myotome heightø The end point of the curves represents the last sample befare the larval group died out. Two groups, However, survivea beyond t.he experimental period and .through metamorphosis .. These were the groups fed live feed All other groups died out during,the experimental period. The typical development in dry weight was a decrease during the first ten days and then either death, stabilization, or increase of dry weight.. The stabilization and also often th,e incre·ase was partly due to the dying out of the smallest l~rvae. This ' . might mask the ~eal results. There is, however, always the starv-ation group and the group fed the standard diet to compare with_ In all experiments the group given the standa~d diet lived langer than the starvation group and had higher dry weight. This indi-cates that the larvae fed the standard.diet were able to utilize energy from ,it ~h~le the starving larvae consurned themselves (Huse 1981}. In no instance, however, did the larvae fed the standard diet grow significantly compared to the start sample. No l~rvae fed the standard diet with hormones added lived langer than larvae fed the standard diet without hormones. On the contrary, where there was a difference in survival .it was alwa~s in favour of the stondard diet. This also was the case with - 6 -growth, althought not so pronounced. The only inert substance to perform hetter than the standard diet was a diet made from cod roe prepared by professor Jan Raa, The University of. Tromsø. The group fed this diet grew significantly compared to the start sarnple, but nevertheless it died out after sixteen days sirnul-tanously with the standard diet group. Both rotifers and living wild plankton gave survival to rneta-rnorphosis, indicating that both larvae and systems were of adequate quality. While we regard the experiments with larval digestive enzyrnes as strictly qualitative, the results nevertheless give an indication that pepsin- and chymotrypsin~ like activities are present in 5 day old unfed larvae. These result therefore support the first of the two hypotheses we wanted to test, that the fish larvae are fully developed at first feeding. However, the. glands may be developed to a stage where enzymatically active protein~ may be produced, while other functions of the digestive system could be far from maturity. Preliminary experiments with young, 'fully developed cod, indicate that the levels of digestive enzymes are reduced to very low lev·els after 4 to 6 weeks of fasting. Intraperitoneal administr,ation of dexarnethasone resulted in significant increases of enzymes in the alirnentary canal after three days. The. development seems, therefore to be to some extent reversible. The lack of succes in the feeding experiment with ·added hormones may have many causes. Even if we have guesse~ the right substances, the number of concentrations we 'have been able to test are few. We-belive that further work along the same line may aid in an-swering the.question: Are external factors needed to continue or to complete the development in young cod larvae? 7 LITTERATURE CITED DOCKRAY, G.J 1979 Comparative biochemistry and physiology ,.gut hormones.. Ann.. Rev.. Physio1 .. , 41:83 - 95 .. FLUCHTERr Jo1982. Substance essential for metamorphosis of fish 1arvae extracted from Artemia.. Aquacu1ture, 27:83 - 85. HUSE, I. and JENSEN, P.. 1980" Status of ~n 'intensive cod rearing project in Norway.. Coun" Meetø int .. Coun. Explor. Sea· 1980 (F:23) .. HUSE, I. 1981 .. An attemp·t to start feed cod larvae with artificial Coun Meet int" Coun .. Exp1or. Sea 1981 (F 14) o HUSE, I. and JENSEN, P. 198la Present, status of an intensive cod larvae rearing experiment at Austevoll. Coun. Meet. int Couno Explor Sea 1981 (F:13). LARSON, L.I. and REHFELD, J.,F. 1977. Evidence for a common evolutionary origin of gastrin and cholecystokinin. Nature, 269:335 - 338e METZLER, D.E. 1977. Biochemistry. The chemical reactions of 1iving ce11s.. Academic Press, New Yotk. RICK., t..Y.. 19 7 4.. Chymotrypsine in H .. U .. i Bergmeyer, ed. Methods of·enzymatic ana1ysis, Vo1.2, Ver1ag Chemie, Weinheim,: 1006- 1012 .. -RICK, W. and FRITSCH, W.P .. 1974. Pepsineø ibid,: 1046- 1057. f-:I: (!) u.: ~ pg 70 • • 1 •• ..,. ...... ___ 2 e ø 3 50· )C•. ~:. •• - -111 •······ ... 40 30 Fig.l. Growth (pg dry weight means) of cod larvae start fed with: l.No food. 2.Standard diet (S). 3.S+Caerulein. 4.8 + Dexamethasone (10mg/500g). 5øS+Dexamethasone (1000mg/500a). ,ug Fig.2 .. / / / / / / / / DAYS AFTER HATCHING / / .~ / / / / • • 1 ·-----. 0-···-··-··· ø 3 ·.....;·-·-· 4 0 0 5 X·········· ·IK 6 Growth (pg dry weight means) of cod larvae start fed with: l.No food 2.Standard diet (S). 3.S+Histamine. 4.Rotifers. S.S+Thyroxine. 6.S+Ecdysone. ,ug 1 ----· 2 •·········· .of( 0-·-·-0 Fig .. 3. Growth (p.g dry weight means) of cod larvae start fed with: lsNO food 2@Standard diet (S) G 3.Squid meal~ 4.S+Zooplankton extract .. • • ·--- --· 0-·-·-0 JC- • - - - • - ·)( Hl Fig .. 4.. Growth (pg dry weight means) of cod larvae start fed with: l.No food.. 2.Standard diet (S). 3&S+Ecdysone+Caerulein~ . Dexamethasone+Thyroxine. 4.Cod roe. l l l l _,'IJ. HIN 1 ·-----· 2 0-·-·-0 3 Fig$ 5. Growth (pg dry weigh·t tneans) of cod larvae start fed with: l No food. 2DStandard diet 3.Living collected zooplanktono 

Endocrine and nutritional factors affecting the first food uptake by cod larvae

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Endocrine and nutritional factors affecting the first food uptake by cod larvae

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