The Israeli Journal of Aquaculture - Bamidgeh, IJA_65.2013.897, 6 pages
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Ontogenetic Changes in the Activity of Chymotrypsin and Carboxypeptidases A and B in Mud Crab, Scylla serrata Augusto E. Serrano, Jr.* Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines in the Visayas, Miagao, Iloilo, Philippines (Received 4.8.12, Accepted 14.12.12) Key words: ontogeny, Scylla serrata larvae, chymotrypsin, carboxypeptidase A, carboxypeptidase B Abstract The ontogenetic pattern of an endopeptidase (chymotrypsin) and two exopeptidases (carboxypeptidases A and B) in larvae of the mud crab, Scylla serrata, are described. Specific activity of chymotrypsin was detected in all larvae stages. The activity was about 25% of the maximum at stage Z1, doubled at Z2 and Z3, declined to 40% at Z4 and Z5, abruptly increased to maximum activity during the megalopa stage, and fell to about 33% at the first crab stage, CI. Carboxypeptidase A activity was low at Z1, gradually increased from 4% to 13%, 19%, and 27% of the maximum at Z5, markedly increased to 68% at the megalopa stage, and finally peaked at CI. Carboxypeptidase B activity started at 9%, declined to 4%, abruptly increased to almost 50% at Z3, remained high at Z4, Z5, and the megalopa stage (50%, 61%, and 50% of the maximum), and finally peaked at CI. The overall changes could be related to changes in diet and feeding habits, or to behavioral, mechanical, and physiological changes, or their combination during development of S. serrata larvae.
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Serrano
Introduction Mud crab is of great economic value and its aquaculture is a major source of income for coastal fishermen in the central part of the Philippines. The increase in aquaculture demand for juveniles has led to a shortage of supply and some municipalities, therefore, ban the export of juvenile crabs. Practically all crablets used in grow-out culture are collected from the wild. A lack of basic information on broodstock biology and larvae physiology has been a major bottleneck in research attempts to develop a stable hatchery technology for this crustacean. Broodstock and spawning technology have been developed in the laboratory (Quinitio et al., 1999) but stable and efficient larvae rearing is yet to be developed. Mortality is high at all stages of development, with a marked peak during the first pre-metamorphic stages (Z5 to megalopa). Digestion in crustaceans such as shrimp involves at least two steps: (a) endopeptidases (i.e., trypsin and chymotrypsin) in the digestive tract and (b) exopeptidases in the cells (Dall and Moriarty, 1983). Acidic peptidases (carboxypeptidases A and B and aminopeptidases) also play important roles in shrimp digestion; 2 h at pH 7.5 followed by 2 h at pH 4.0 resulted in complete breakdown of protein in an in vivo study of these peptidases from grass shrimp (Lan and Pan, 1993). Acidic exopeptidases generally exist in lysosomes. Their role in digestion agrees with microscopic observations of intracellular digestion in B cells of the shrimp hepatopancreas (Al-Mohanna and Nott, 1986). Understanding protein digestion is relevant for many steps in shrimp production. The life history of crustaceans is marked by changes in morphology and behavior, with shifts in feed preferences and adoption of a benthic existence in larvae stages. Such ontogenetic events are accompanied by changes in metabolic rates and digestive enzyme activity (Lemos et al., 1999). The characterization and quantification of proteolytic enzyme activity in different development stages of crustaceans may contribute to improving feeding conditions during the entire production process. At very early stages of development, crustacean larvae may be ill-equipped to digest food materials and rely on enzymes in live food organisms to assist digestion (Kumlu, 1999). Enzymes in live prey are released into the digestive tract of crustacean larvae by autolysis or zymogens that activate endogenous enzymes within the larvae gut (Kumlu and Jones, 1995). However, contrary to this hypothesis, high levels of enzyme activity have been detected in penaeid larvae right after hatch (Jones et al., 1991). In mud crab larvae (S. serrata and S. paramamosain), the specific activity of protease, amylase, cellulase, and lipase varies with development stage (Hong et al., 1995). The ontogenetic patterns of an endopeptidase activity (trypsin) and an aminopeptidase activity (leucine aminopeptidase; LAP) in Scylla serrata have been described (Serrano and Traifalgar, 2012). The activity of trypsin was elevated in stage Z2, remained low in later stages, rose again from the megalopa stage, and reached the highest activity in the first crab stage (C1). In contrast, LAP enzyme activity increased gradually from stage Z3 and reached a maximum at stage C1. The present study characterizes the ontogenetic pattern of another endopeptidase (chymotrypsin) and two exopeptidases (carboxypeptidases A and B) in Scylla serrata larvae, megalopae, and first crab instar. Materials and Methods Experimental animals and live food. Crab larvae were produced as described by Serrano and Traifalgar (2012). Briefly, broodstocks were cleaned and disinfected in 100 mg/l formalin bath for 30 min, reared individually on sandy substrates in a flow-through water system, and fed squid (Loligo spp.) and fresh mussel meat (Perna viridis Linnaeus, 1758) ad libitum. Berried females were transferred to incubation tanks and not fed during the incubation period. Eggs were sampled and examined for development. Following hatching, zoea in the strongly phototactic first stage Z1 were collected using a plankton net and transferred to experimental rearing tanks. Rotifers were maintained and propagated in fiberglass tanks, fed green algae Tetraselmis chuii or marine Chlorella, and harvested by filtration through 30-µm mesh plankton nets. Appropriate-sized rotifers were fed to the crab larvae. Also, commercially available Artemia cysts were hatched in the laboratory following manufacturer instructions and fed to the S. serrata larvae.
Ontogeny of chymotrypsin, carboxypeptidase A and B activity in mud crab larvae
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Enzyme extract preparation. At every stage, larvae were seined and concentrated using a 0.5-mm mesh screen, thoroughly washed with sea water, and prepared for enzyme extraction. The larvae were washed with a cold extraction solution of 50mM citrate phosphate buffer (pH 7.0), weighed, and homogenized in the same solution at a ratio of 1:20 (wet tissue wt:volume) in an Ultraturrax homogenizer. The homogenate was centrifuged at 4000 rpm for 15 min and the supernatant was filtered and used for enzyme preparation. Enzyme assay. Assays were carried out at 25°C and values are means of triplicate estimates±standard error of the mean (SEM). Assays were conducted with appropriate controls, including non-enzymatic hydrolysis. Protein contents were measured by the method of Bradford (1976), using bovine serum albumin as the standard. Chymotrypsin was determined using the method of Hummel (1959). Briefly, the assay mixture consisted of 1.4 ml 1.07 mM benzoyl-L-tyrosine ethyl ester (BTEE) dissolved in 50% (w/w) methanol, 1.0 ml 80mM Tris-HCl buffer (pH 7.8) containing 0.1 M CaCl2, and 0.3 ml extract in a final volume of 2.7 ml. The reaction was stopped by adding 0.3 ml 30% acetic acid. The hydrolysis of N-benzoyl-L-tyrosine ethyl ester into Nbenzoyl-L-tyrosine + ethanol causes an increase in absorbance at 256 nm. Enzyme activity was expressed as μmol BTEE produced/min/mg protein at 25°C and pH 7.8. Carboxypeptidase A was assayed by the method of Folk and Schirmer (1963) as modified by Appel (1974). The assay system consisted of 1.0 ml 25mM Tris-HCl buffer (pH 7.5) containing 0.5M NaCl, 1.4 ml 1.0mM hippuryl-L-phenylalanine dissolved in buffer, and 0.3 ml enzyme extract in a final volume of 2.7 ml. The reaction was stopped by adding 0.3 ml 30% acetic acid. The rate of hydrolysis of hippuryl-L-phenylalanine was determined by measuring the increase in absorbance at 254 nm. Enzyme activity was expressed as the increase in absorbance due to the formation of hippuric acid/min/mg protein at pH 7.5 and 25°C. Carboxypeptidase B was assayed by the method of Folk et al. (1960) as modified by Appel (1974). The assay system consisted of 1.0 ml 25mM Tris-HCl buffer (pH 7.7) containing 0.1 M NaCl, 1.4 ml 0.1mM hippuryl-l-arginine dissolved in buffer, and 0.3 ml crude enzyme. The reaction was stopped by adding 0.3 ml 30% acetic acid and optical density was measured at 254 nm. Enzyme activity was expressed as the increase in absorbance resulting from the formation of hippuric acid as the product of the hydrolysis of hippuryl-L-arginine/min/mg protein at 25°C and pH 7.7. Statistical analysis. Data were tested for homogeneity of variances and normal distribution, then subjected to one-way analysis of variance (ANOVA) at α = 0.05 to determine if differences between treatments exist (Zar, 1984). Differences were further tested by Tukey’s Post-hoc test. Results Activity of all three enzymes was detected at all larval stages (Fig. 1).
Chymotrypsin
b
Carboxypeptidase A
2.5 2.0 1.5 1.0 0.5 0
c
Carboxypeptidase B
Hippuryl-Larginine/min/mg protein
80
Hippuric acid/min/mg protein
BTEE produced/min/mg protein
a
70 60 50 40 30 20 10 0
Development stage
Development stage
Development stage
Fig. 1. Changes in specific activity of (a) chymotrypsin, (b) carboxypeptidase A, and (c) carboxypeptidase B in mud clam, Scylla serrata, during larvae development from zoea stages 1-5, through the megalopa stage (MG), and in the first crab stage (C1). Error bars indicate standard errors of means. Means with different superscripts differ significantly (p
