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Changes of digestive enzymes during growth of cultured juvenile


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									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.


         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.

        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: koueta@ibba.unicaen.fi


         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 semi-
closed 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
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
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.
9- Statistical analysis
        The results obtained were compared between groups using Anova test


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).
          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.

       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.


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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.
      Figure I. Non   specific     protmtytic activity


O,O            I               I            I            I.

          0           10              2.0       a0
                           Time (days)





    I-            .
    ,-                \
     ki                       \


Fiqure 6. Effect of ration on chymotrvpsin activity

        - * - Ration 20% d-Ration 40% (b) -d-Ad libiim (c)
    I          \
I   4   I            8

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