Sepia officinalis

ICES Annual Science Conference in Brugge, Belgium ( 27 - 30 September 2000) Paper code: ICES CM 2000/O: 06 Theme session: Sustainable Aquaculture Development. Changes of digestive enzymes during growth of cultured juvenile cuttlefish Sepia officinalis L. ( Mollusca Cephalopoda ). Effect of enriched diet and ration. Koueta, N., Le Cal&, A., Noel, B. and Boucaud-Camou, E.

Abstract. The culture of cephalopods is becoming an interesting area due to their fast growth their scientific importance, and to their commercial value. Juvenile as well as mature cuttlefish are characterized by a carnivorous diet, but only mature animals are been reared using artificial diet. The use of artificial diet to rear juvenile cuttlefish is still difficult. In order to formulate an artificial diet well accepted by juvenile cuttlefish, their digestive capability was studied Biochemical estimation of temporal development of digestive enzymes in juvenile cuttlefish Sepia oficinalis shows a correlation between growth and proteolytic activities from hatching to 30 days old. However, trypsin activity increases during the first 15 days then decreases. Chymotrypsin activity increases during 30 days. After hatchling, the group of juvenile cuttlefish fed on enriched PUFA (polyunsaturated fatty acids) shows a higher level of trypsin activity than the group fed on live prey. During 20 days the level of chymotrypsin activity depend on food quality. Digestive enzymes present different levels with the rations used.

Keys words. Diet - digestive - enzymes - growth -juvenile cuttlefish - rearing. Addresses. Koueta, N., Boucaud-Camou, E. and Le Calve, A. : Laboratoire de Biologie et Biotechnologies Marines, UniversitC de Caen, 14032 Caen, France. Noel, B. : Dielen Laboratoires, port des Flamands, 50110 Tourlaville, France. Correspondence to N. Koueta: tel: 33 231 56 55 96; fax 33 231 56 53 46; e-mail: [email protected]

Introduction Juvenile cuttlefish as mature are characterized by a carnivorous diet. Many investigations for mature cuttlefish rearing using altenative diets have been made (Richard, 1971 et 1975; Boletzky, 1989; Koueta and Boucaud-Camou, 1999) but for young animal it is still difficult to use artificial diet during the first month of their live DeRusha et al., 1989; Castro, 1991; Castro et al., 1993). The young animal remains fragile and grows less than with live prey. To resolve this problem, it necessary to formulate artificial diet well accepted by juvenile cuttlefish. In this way , many diets are been tested to enhance survival and growth of juvenile cuttlefish during the first days of rearing, but the optimisation of alternative or

artificial diet depends essentially to quality and quantity of digestive enzymes and their physiological regulation during the juvenile phase. Previous investigations realized by Boucaud-Camou (1973), Boucher-Rodoni (1983) have shown the presence of many digestive enzymes in digestive tract of mature cuttlefish. So non specific proteolitic activity, trypsin activity, chymotrypsin activity, amylasic activity and phosphatase activity have been detected. Boucaud-Camou and Roper (1995) have detected many digestive enzymes like protease, chymotrypsin phosphatase but no trypsin and amylase in Octopodidae, Bolitaenidae, Ommastrephidae and Enoploteuthidae during their plantonic post-larval phase. Yim (1978) and Yim and Boucaud-Camou (1980) have shown that the digestive gland of juvenile cuttlefish is quite cytological different to the one of mature animal. The differentiation to mature form appears during the first 30 days of the juvenile live, but the digestive enzymes produce during this juvenile phase have not been studied. The aim of this work was to study the changes of digestive enzymes during juvenile cuttlefish growth and the effect of quality and quantity of diet on their digestive capability. Materials and Methods. I- Experimental animals. All the eggs were laid in the laboratory by females trawled off the Normandy coast and maintained in a large tank receiving water from the sea. The eggs were placed on floating sieves distributed in tanks connected to the semiclosed system as previously described (Koueta and Boucaud-Camou, 1999). As hatching lasted several days, hatchlings were placed in small tanks of 707 cm2 in groups of ten animals. 2- Rearing system. The culture, filtration, water circulation, and water oxygenation systems, the light and temperature conditions, and the stocking and culture densities were as previously described (Koueta and Boucaud-Camou, 1999). The pH of the seawater was 8, salinity 35.5%, and the concentration of 02 measured with an electronic oxygenmeter was optimal, (8.9-9.7 ppm corresponding to a saturating rate of 101 to 110%). NH4+ measurements made with a calorimetric kit revealed < 0.5mg1, and hence the NH3 concentration was < 0.02 mg/l at the pH of rearing. For nitrites and nitrates the concentrations measured with a calorimetric kit were ~0.1 mg/l and < 10 mg/l respectively. Physical and chemical parameters were maintained constant by continual renewal of oxygenated seawater, by avoiding surpopulation, and by removing dead cuttlefish, dead prey, and food remains. Before entering the system the natural seawater which was used to renew the circuit ran through a system of U.V. lamps with a flow rate of 60 l/h (93% renewal per day). The mechanical filters, which consist of foam and synthetic fibres, were cleaned daily with seawater (to avoid lethal osmotic shock to the nitrite bacteria). The temperature of seawater was maintained between 19.5”C - 20.5”C by the heating elements in the conditioning tank. The rearing device received 12 h of light/ 24 h. 2- Experiment I A total of 60 juvenile cuttlefish were selected, measured, weighed, and randomly ditributed in small tanks. Each tank contained four animals well separated by a thick partition. The cuttlefish were divided into 2 groups of 30 animals receiving respectively adult artemia and artemia enriched with Gabolysat during 10 days then Crangon crangon were offered during 20 days. The juvenile cuttlefish were fed ad libitum. 3 - Experiment 2

A total of 90 juvenile cuttlefish were selected, treated as previously described, and divided into 3 groups of 30 animals receiving respectively 20% and 40% of their body weigth of young shrimps (Crangon crangon) and ad lib&urn feeding. 4- Fish oil and Gabolysat. The Gabolysat was provided by Dielen Laboratoires and contained respectively 74% of proteins, 9% of lipids containing 12.6% EPA and 21% DHA of total fatty acids for the Gabolysat. 5- Feeding methodology According to the diet previously described, the preys were offered once each day at 10 a.m. The daily ration (maximum ration) for juvenile cuttlefish as observed by Koueta and Boucaud-Camou (in preparation) was 40 % of animal body weight. The daily ration was adjusted according to animal weight after 5 or 10 days during 30 days of rearing 6- Sample preparation Juvenile cuttlefish were weighed and measured at the end of the experiment, killed by immersion in liquid nitrogen and finally stored at -80 “C until analysis. The samples were homogenized with chilled buffer (lmV60mg of tissue) containing Tris O.lM EDTA 3 n&I, boric acid 0.08M and 10% of glycerol (Koueta 1983), then centrifuged for 1 h at 10 000 g, 4°C. 8 Erqme assays For non specific proteolitic activity the protocol used was based on that described by Rinderknecht et al (1968) using “Hide Poder Azur” as substrate. Trypsin activity was detected using Tsunematsu et al (1985) method with Z-Arg-p-Na (ZAPA) as substrate. Chymotrypsin activity was measured as Delmar et al.( 1979) using SAAPPNA (Succinyl-Ala-Ala-Pro-Phe-p Nitroanilide) as substrate. Amylase activity was tested using Sigma kit no 577-20. The optic density was measured with a spectrophotometer SECOMAM PRIM. For the quantification of proteins, the assay followed the method of Lowry et al. (1951) 9- Statistical analysis The results obtained were compared between groups using Anova test Results Non spec@proteolytic activity figure 1) Proteolytic activity increase during the rearing and does not depend on the quality of the food offered. The activity is 5 times higher after 30 days than at first day of rearing. Trypsin activity figure 2) Trypsin activity changes during growth of juvenile cuttlefish. This enzyme activity increases greatly after 10 days of rearing then remains stable until 30 days .Enriched diet stimulates the production during 20 days. Chymotrypsin activity figure 3) The chymotrypsin activity increases during 30 days. Enriched diet stimulates the production during 20 days. Amylase activity figure 4). The amylase activity is detected after 10 days of rearing then increases greatly after 20 days. This activity decreases at the end of the experimental rearing at 30 days. The quality of the food offered has no effect on the secretion. Eflect of the rations on the digestive exymes. For trypsin , chymotrypsin and amylase activity, the secretion is better between 20 and 30 days for the juvenile cuttlefish receiving 40% of their body weight in food or fed ad Zibitum. (figure $6, and 7).

Discussion Enriched diet does not increase secretion of non specific proteolytic enzymes &ring juvenile cuttlefish growth, but trypsin and chymotrypsin activities depend on the quality of the food during 20 days of rearing. Cahu and Zambonino-Infante (1994, 1995); Peres et al. (1996) have shown that in carnivorous fish than Dicentrarchus labrax trypin and chymotrypsin activities increase greatly after hatching and these secretions change when protein increases in the diet. In juvenile cuttlefish we observe the same phenomen during 20 days of rearing. In our previous work (Koueta et al. 2000) we have suggested some biochemical indices for instantaneous growth estimation in juvenile cuttlefish. The increase of non specifis proteolitic activity during rearing also suggests that this digestive activity could be used as biochemical indice for juvenile cuttlefish growth as Aspartate transcarbamylase activity and used to predict the effect of biotic factors on growth and recruitment in youg cephalopods collected in field. Boucaud-Camou and Roper 1995,1998 have not detected amylasic activityin plantonic post larve of some cephalopods. This investigation confirms the absence of this enzyme during the10 first days of rearing. Cahu and Zambonino-Infante (1994, 1995) in larves of carnivorous fish Dicentrarchus labrax as Ribeiro et al. (1999) in larvae of Solea senegalensis observe that amylasic activity decreases during growth. In juvenile cuttlefish this activity increases. Boucaud-Camou (1973) and Yim (1978) have detected amylase activity in mature cuttlefish. This evolution of amylase activity could be specific to cephalopods development. The great increase of non specific proteolytic enzymes during the experiment could be due to pepsin and cathepsin activities as suggested Morishita (1972) in Octopus vulgaris. This investigation shows that the ration affects the digestive enzymes secretion. The maximum ration, 40% of the animal body weigth during the first month (Koueta and Boucaud-Camou in preparation) is necessary for a better secretion. The trypsin and chymotrypsin activity decrease when juvenile cuttlefish are under fed. In previous investigations we have shown that enriched diet increase survival and growth of juvenile cuttlefish and they are able to accept frozen prey after 10 days of rearing. This investigation confirms our previous result because the enriched diet stimulates trypin and chymotrypsin activity greatly during 10 days. This change induce the best digestive capacity of juvenile cuttlefish at 10 days old. Others previous works (Hjelmeland et al. 1984; Baragi and Love11,1986) have suggested that good assimilation and digestion of the food are due to an increase of trypsin activity The presence of enzymatic capacity before feeding suggests that this digestive enzymes are not induced by the food. But the enriched diet could stimulate the maturation of digestive tract and digestive organ then increasing secretion of digestive enzymes. In our further investigations the effect of enriched diet on the ultrastructural development of digestive tract and digestive organ during growth of juvenile cuttlefish would be carried up. Acknowledgements. This work was supported by the Conseil Regional de Basse Normandie and Dielen Laboratoires , the rearing was done in CREC at Luc sur mer. We thank I. Probert for his help in English. References Baragi, V. and Lovel, R.T., 1986. Digestive enzyme activities in striped bass from first feeding through larvae development. Trans.Am. Fish. Sot., 115 : 478484.

Boletzky, S.V., 1989. Elevage de Cephalopodes en aquarium: acquis recents. Bull Sot Zoo1 Fr 114: 57-66. Boucaud-Camou E., 1973. Etude de l’appareil digestif de Sepia oficinalis L. (Mollusque : Cephalopode). Essai d’analyse experimentale des phenomenes digestifs. These doctorat d’etat es science, Universite Caen. 208 p. Boucaud-Camou E., 1974. Localisation d’activites enzymatiques impliquees dans la digestion chez Sepia o&inaIis L. Arch. Zool. Exp. Gen. 115: 5-27. Boucaud-Camou E. and Roper C.F.E., 1995. Digestive enzymes in paralarval Cephalopods. Bull. Mar. Sci. 57: 313-327. Boucher-Rodoni R.,1981. Etude de la glande digestive de deux Cephalopodes, au cours de la digestion et au tours du cycle de vie. These doctorat d’etat es science, Universite Paris sud, centre d’ Orsay, 176~. Cahu C.L. and Zambonino Infante J.L., 1994. Early weaning of sea bass (Dicentrarchus Zabrax) larvae with a compound diet : effect on digestive enzymes. Comp. Biochem. Physiol. 109A: 213-222. Cahu C.L. and Zambonino Infante J.L., 1995. Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus 1abra.x) : effect of weaning with different protein sources. Fish Physiol. Biochem. 14: 43 l-437. Castro, B.G., 199 1. Can Sepia oflcinalis L. be reared on artificial food ? Mar Beh and Physiol 19: 83-86. Castro, B.G., Di Marco, F.P., DeRusha, R.H. and Lee, P.G., 1993. The effects of surimi and pelleted diets on the laboratory survival, growth, and feeding rate of the cuttlefish Sepia ofjcinalis L. J. Exp.. Mar. Biol. and Ecol. 170: 24 l-252. DeRusha, R.H., Forsythe, J.H., DiMarco, F.P. and. Hanlon, R.T., 1989. Alternative diets for maintaining and rearing cephalopods in captivity. Lab. Ani. SC. 4,306-3 12. Delmar E.G., Largman C., Brodrick J.W. and Geokas M.C., 1979. A sensitive new substrate for chymotrypsin. Analyt. Biochem. 99: 3 16-320. Hjelmeland, K. Huse, J.T. and Molvik, R.J. 1984. Trypsin and trypsinogen as indices of growth and survival potential of cod (G..morhua) larvae. In E. Dahl, D.S. Danielsen, E. Morkness and Solemdal Editors), The propagation of Cod (G. morhua). Part 1. Institute of Marine Research, Flodevigen Biological Station, pp. 183-2 11.. Koueta N., 1983. Contribution a l’etude de la glande salivaire posterieure de Cephalopodes Decapodes. These doctorat 3”*‘: cycle, Universite Caen, 69 p. Koueta N., Castro B.G. and Boucaud-Camou E., 2000. Biochemical indices for instantaneous growth estimation in young Cephalopod Sepia o$fkinaEis L. J. Mar. Sci. ICES 57: l-7.

Koueta N. and Boucaud-Camou E., 1999. Food intake and growth in reared early juvenile cuttlefish Sepia oficinalis L. (Mollusca : Cephalopoda). J. Exp. Mar. Biol. Ecol. 240: 93-109. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Morishita T., 1972. On the existence of cathepsins A, B and C in Octopus liver. Bull. Jpn. Sot. Sci. Fish. 38: 1057-1060. Peres A., Cahu C.L., Zambonino Infante J.L., Le Gall M.M. and Quazuguel P., 1996. Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae. Fish Physiol. Biochem. 15: 237-242. Ribeiro L., Zambonino-Infante J.L., Cahu C. and Dinis M.T., 1999. Development of enzymes in larvae of Solea senegalensis, Kaup 1858. Aquaculture 179: 465-473. Richard, A., 1971. Contribution a l’etude experimentale de la croissance et de la maturation sexuelle de Sepia oficinalis L. (Mollusque : Cephalopode). These de Doctorat es Sciences Naturelles, Universite de Lille. Richard, A., 1975. L’elevage de la seiche (Sepia oficinalis L., Mollusque).lO* European Symposium on Marine Biology, Ostend, Belgium, pp. 359-380. Rinderknecht H., Geokas M.C., Silverman P. and Haverback B.J., 1968. A new ultrasensitive method for the determination of proteolytic activity. Clin. Chem. Acta 21: 197-203. Tsunematsu H., Nishimura H., Mizusaki K. and Makisumi S., 1985. Kinetics of hydrolysis of amide and anilide substrates of p-Guanidino-L-Phenylalanine by bovine and porcine trypsins. J. Biochem. 97: 6 17-623. Yim M., 1978. Developpement post-embryonnaire de la glande digestive de Sepia oficinalis L. (Mollusque, CCphalopode).These doctorat 3eme cycle, Universite Caen, 75 p. Yim M. et Boucaud-Camou E., 1980. Etude cytologique du developpement postembryonnaire de la glande digestive de Sepia oficinalis L. Mollusque Cephalopode. Arch. Ana. Mic. Morph. Exp. 69: 59-79.

Legends of figures Figure 1. Non specific proteolytic activity during juvenile cuttlefish rearing. Effect of the quality of the diet. Figure 2. Trypsin activity during juvenile cuttlefish rearing : effect of different diets. Figure 3. Change of chymotrypsin activity during juvenile cuttlefish rearing : effect of different diets. Figure 4. Effect of the quality of diet on Amylase activity of juvenile cuttlefish. Figure 5. Effect of ration on trypsin activity of juvenile cuttlefish. Figure 6. Effect of ration on chymotrypsin activity of juvenile cuttlefish. Figure 7 Effect of ration on Amylasic activity of juvenile cuttlefish.

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