Characterization of chymotrypsin activity during early ontogeny of larval red drum (Sciaenops ocellatus)

The temporal evolution of chymotrypsin activity during early ontogeny of laboratory reared red drum larvae was accomplished using a combination of biochemical assays and electrophoretic methods (substrate SDS-PAGE). Optimal functional conditions for chymotrypsin were also determined. Chymotrypsin activity was first detected prior to the onset of exogenous feeding. Total chymotrypsin activity increased with age and standard length. Specific activity was greatest on day 10 post-hatch. Maximal chymotrypsin activity was observed at 50 °C, pH 7.8, and Ca²⁺ concentration of 25 mM. Using substrate gel electrophoresis and specific inhibitors the molecular weight of red drum chymotrypsin was estimated to be 26–27 kD. Our results indicate that the digestive system of red drum larvae is capable of alkaline proteolysis before first feeding and suggest that chymotrypsin may have potential as an indicator of nutritional condition.     

抗裂与易裂枣内源激素含量和细胞壁代谢相关酶活性比较

 以枣抗裂果品种‘圆铃枣’和易裂果品种‘俊枣’为材料,测定了果实生长发育曲线、果 形指数及种子败育率,并对果皮、果肉、种子中内源激素含量及果皮、果肉中细胞壁代谢相关酶的活性 进行了检测。结果表明,‘俊枣’果形指数和种子败育率均显著高于‘圆铃枣’。果实发育后期‘俊枣’ 果皮中的GA3 含量、果肉中的IAA 含量明显高于‘圆铃枣’,而‘圆铃枣’果肉及种子中的ABA 含量高 于‘俊枣’;‘俊枣’果肉及种子中（GA3 + IAA + ZT）/ABA 的比值在整个果实生长发育期均高于‘圆铃 枣’。果实生长发育后期‘俊枣’果皮中的果胶酶及纤维素酶活性高于‘圆铃枣’,且‘俊枣’果肉中的 POD 及PPO 活性也较高。以上结果显示,枣果实生长发育后期易裂品种‘俊枣’果肉中的IAA 积累较多, 而抗裂品种‘圆铃枣’果肉及种子中ABA 含量显著高于易裂品种;易裂品种‘俊枣’果肉及种子中（GA3 + IAA + ZT）/ABA 的比值较高,果皮中的果胶酶、纤维素酶活性影响裂果的发生,其POD 及PPO 活性相 对较高。     

海洋贝类体液防御机制的研究进展

近几年，随着人们将海洋贝类作为食物的兴趣日益浓厚，科学家们对贝类及其防御机制的研究也越来越深入，对它们在生态、社会、经济、营养等方面的作用以及它们的致病微生物的特性进行了大量研究。双壳贝类由于其滤食习性，使它们体内蓄积了大量细菌，成为人类致病菌的积极携带者。因此，研究贝类与微生物之间的相互作用对维护人类健康有重要意义。     

Effects of in vitro exposure to ozone and/or hyperoxia on superoxide dismutase, catalase, glutathione and lipid peroxidation in red blood cells and plasma of rainbow trout, Oncorhynchus mykiss (Walbaum)

In aquaculture, ozone is used as a disinfectant. In its production, extensive amounts of oxygen are formed resulting in hyperoxic conditions in culture units. Both ozone and hyperoxia have the potential to be toxic via pro‐oxidant mechanisms and to activate antioxidant defence systems in cultured species. To eliminate systemic effects, blood of rainbow trout, Oncorhynchus mykiss (Walbaum), was exposed in vitro for 5 min to ozone/hyperoxia or hyperoxia, and changes in antioxidant defences and lipid peroxidation were measured after exposure. Ozone exposure caused severe damage in red blood cells (rbc) detected as increased lipid peroxidation and oxidized glutathione (GSSG) levels in both plasma and rbc. Oxygen exposure alone increased intracellular lipid peroxidation and GSSG levels 10 min after exposure and was not evident in the plasma at any time. Ozone, but not oxygen exposure, decreased reduced glutathione (GSH) levels in plasma, and the changes were negatively correlated with increased lipid peroxidation in rbc, indicating that extracellular GSH has a dynamic role in the protection of rbc from direct oxidation by ozone. Both ozone and hyperoxic conditions increased superoxide dismutase (SOD) activity in rbc 3 and 6 h after exposure. In contrast, catalase activity was only increased 10 min after oxygen exposure, suggesting other catalase activation mechanisms rather than enzyme induction. The recovery of lipid peroxidation and GSSG levels in rbc after hyperoxia, but not ozone exposure, indicated a capacity to defend against hyperoxia‐produced oxidative damage, but an overwhelming of antioxidant defences by ozone in rainbow trout rbc in vitro.     

Digestive enzyme activity of the red porgy (Pagrus pagrus, L.) during larval development under culture conditions

The ontogenic development of the main digestive enzymes (proteases, amylase and lipase) in the red porgy, Pagrus pagrus, larvae was assayed during the larval development. The green water technique was carried out for larval rearing and whole‐body homogenates were used for enzymatic assays in triplicate. Significant alterations in specific activities of all digestive enzymes measured during the period of this study were mostly related to metamorphosis and weaning. Trypsin‐ and chymotrypsin‐specific activities were first detected on day 3, together with opening of the mouth, and slightly increased until 25 days after hatching (DAH). After this period, the specific activities of these enzymes slightly decreased. Pepsin was first detected on day 28, concurrent with stomach formation, and a sharp increase was observed until 30 DAH. A slight decrease was measured from this date until the end of the experiment. Both amylase and lipase were measured for the first time on days 2 and 4 respectively, and the specific activities of these enzymes showed similar patterns during the first week of the study. Then, slight variations were observed until 30 DAH and while lipase‐specific activity declined, an increase in the specific activity of amylase was found until the end of the experiment. It is concluded that the variations observed in the specific activity of digestive enzymes were related to either metamorphosis, such as the formation of the stomach (28 DAH), or to changes in food composition. The profile of the developmental pattern of the main digestive enzymes detected in P. pagrus is similar to that described for other Sparid species.     

Effects of illumination on early life development and digestive enzyme activities in common pandora Pagellus erythrinus L. larvae

The activities of main digestive enzyme (proteases, amylase and lipase) and animal husbandry (mainly growth and survival) were studied in common pandora Pagellus erythrinus larvae until 30 days after hatching (DAH). Three different illumination levels (10, 30 and 100 lx) were compared in triplicate and green water technique was carried out. At the end of the experiment, illumination did not affect neither survival nor growth except 10 lx treatment. Similarly, specific growth rate (SGR) was different in 10 lx treatment (p < 0.05), although no differences were found in group 30 and 100 lx treatments (p > 0.05). In all groups, trypsin and chymotrypsin specific activities were firstly detected on day 3 related with mouth opening and slightly increased until 20 and 25 DAH respectively, and after this date specific activities of those decreased. Although, there was a significant difference between 10 lx treatment and other experimental groups (p < 0.05), no differences were found in other treatments (p > 0.05). Pepsin was firstly detected on day 25 related with stomach formation and sharp increase was observed until 30 DAH and then slight decrease was measured from this date and no differences were found between all groups. Amylase was firstly determined on day 2 and increased to day 5. After this date, slight decreases were measured in all groups and continued until end of experiments. The highest specific activity of amylase was determined in 30 lx treatment and no significant differences were found between groups (p > 0.05). Lipase was firstly detected on day 4 and increased to day 10. Then, activity of lipase decreased until day 15 and increased again until 25 DAH. Slight decreases were found in all groups until day 30 and continued to end of experiments. No significant differences were detected among groups (p > 0.05).

Finally, the significant improvement in survival, larval development and specific enzyme activities of larvae were determined in 30 lx treated group. It is thought that this phenomenon is related to optimal keeping conditions provided by the medium illumination level for Pagellus erythrinus larvae.     

Stimulating effects of pond water on digestive enzyme activity in the Pacific white shrimp, Litopenaeus vannamei (Boone)

Crude enzyme extracts were obtained from the digestive glands of Pacific white shrimp, Litopenaeus vannamei (Boone), reared in oligotrophic well water and eutrophic shrimp pond water to compare digestive enzyme activity between the two groups. Specific activities of serine protease, collagenase, amylase, cellulase, lipase and acid phosphatase were significantly higher (P < 0.01) in pond water‐reared shrimp (PW shrimp) than in well water reared‐shrimp (WW shrimp). For most enzymes assayed, specific activity was more than two times higher in PW shrimp, and cellulase activity was over six times higher. In contrast, chitinase activity was significantly higher (P < 0.001) in WW shrimp. Higher specific activity of most digestive enzymes in PW shrimp was probably due to natural productivity in the pond water that served as a source of organic substrates, and this increased activity may contribute to the growth‐enhancing effect of shrimp pond water.     

Effect of dietary carbohydrate-to-lipid ratios on growth, lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis, Kaup)

A study was undertaken to determine the effect of various dietary carbohydrate‐to‐lipid ratios on growth performance, whole‐body composition and tissue lipid content in Senegalese sole (Solea senegalensis) juveniles. Data on the dietary regulation of key hepatic enzymes of the lipogenic and glycolytic pathways (glucose‐6‐phosphate dehydrogenase, G6PD; malic enzyme, ME; fatty acid synthetase, FAS; pyruvate kinase, PK and glucokinase, GK) were also generated. Four isonitrogenous (crude protein: 52% dry matter (DM)) diets were formulated to contain one of two lipid levels (11% and 21% DM). Within each dietary lipid level, the nature of the carbohydrate fraction (raw or extruded peas) was varied. Triplicate groups of 54 sole (initial body weight: 23.6±1.2 g) were grown in recirculated seawater over 67 days. Fish were fed using automated feeders. At the end of the study, whole‐body, liver, viscera and muscle samples were withdrawn for analyses. During the experimental period, the mean fish weight about doubled in all treatments. No significant differences were found in growth performance (ranging from 1.1% to 1.4% body weight day^?1) among dietary treatments. High‐fat diets increased whole‐body fat content. Similarly, daily fat gain ranged from 0.54 to 0.78 g kg^?1 day^?1 and highest values were found in fish fed high‐lipid diets. Dietary treatments also affected tissue lipid content (liver, viscera and muscle), with highest values in fish fed high‐fat diets. The nature of dietary carbohydrates had little influence on performance criteria, but affected tissue lipid deposition. The activities of G6PD, ME and FAS were depressed by elevated levels of dietary lipid, confirming the inhibitory effect of dietary fats on lipid biosynthesis. At both dietary lipid levels, ME and FAS activities were little affected by dietary carbohydrate. Activities of PK and GK were not affected by the starch level of the diets. In Senegalese sole juveniles, the lipogenic pathway is more susceptible to modulation by dietary means (particularly through lipid intake) than the glycolytic pathway.     

Trypsin enzyme activity during larval development of Litopenaeus vannamei (Boone) fed on live feeds

Larval stages of the Pacific white shrimp, Litopenaeus vannamei (Boone) were fed standard live diets of mixed microalgae from the first to the third protozoea (PZ1 to PZ3), followed by Artemia nauplii until post‐larvae 1 (PL1). Trypsin enzyme activity for each larval stage was determined using N‐α‐p‐toluenesulphonyl‐l ‐arginine methyl ester (TAME) as a substrate. Results were expressed as enzyme content to assess ontogenetic changes during larval development. Tissue trypsin content (IU µg^?1 DW for each larval stage) was significantly highest at the PZ1 stage and declined through subsequent stages to PL1. This contrasts with previously observed patterns of trypsin development in Litopenaeus setiferus (Linnaeus) and other penaeid genera, which exhibit a peak in trypsin activity at the third protozoea/first mysis (PZ3/M1) larval stage. Litopenaeus vannamei larvae transferred to a diet of Artemia at the beginning of the second protozoea (PZ2) stage were significantly heavier on reaching the first mysis stage (M1) than those fed algae, while survival was not significantly different between treatments. At both PZ2 and PZ3 stages, trypsin content in larvae feeding on Artemia was significantly lower than in those feeding on algae. The rapid decline in trypsin content from PZ1 and the flexible enzyme response from PZ2 suggest that L. vannamei is physiologically adapted to transfer to a more carnivorous diet during the mid‐protozoeal stages.