Mechanism Of Dietary Lecithin Affecting Vitellogenesis Of Red Claw Crayfish, Cherax Quadricarinatus

The redclaw crayfish, Cherax quadricarinatus, is a popular commercial crustacean species in several countries around the world because of their potential large size, fast growing, tolerance to a broad range of temperatures and water quality conditions, and large market potential. Since its introduction to China from Australia in1992, it has not formed a large scale cultivation although some scientific researches have done. The main limitation is small-scale larvae supply which was mainly resulted by mature synchronization rate, low rate of spawning and hatching and livability. At present, many studies have focused on the juvenile redclaw’s growth, survival and body composition, but few have evaluated the broodstock reproduction. As broodstock nutrition affects egg quality, embryonic development and postlarval quality in crustaceans, an evaluation of ovarian maturation will be helpful in understanding the nutrition requirements of the female redclaw during vitellogenesis. In this study, we tried to understand the mechanism of dietary lecithin affecting Cherax quadricarinatus gonadal development based on nutrition, biochemistry, electron microscope and molecular biology methods. The data obtained would provide valuable information to a better understanding on vitellogenesis of crustaceans. It is also helpful to redclaw’s large-scale commercial aquacculture.1. Effects of dietary soybean lecithin on growth and biochemical changes of hepatopancreas and ovary in the female redclaw crayfish, Cherax quadricarinatus.The effects of increasing levels of dietary de-oiled soybean lecithin (SL) on growth and biochemical changes of hepatopancreas and ovary during gonadal development of female redclaw crayfish, Cherax quadricarinatus, was investigated. Five practical formulated diets were supplemented with0%(Diet1),1%(Diet2),2%(Diet3),4%(Diet4) and6%(Diet5) SL. Crayfish (initial weight:25.64±1.53g, eight shrimp per tank) were fed each diet in four replicates for8weeks. Shrimp survival and relative weight gain were not significantly different among the five treatments(P>0.05). Crayfish that were fed diets containing≥2%SL had a significantly higher gonadosomatic index (GSI) than those fed with Diets1and2(P <0.05), while hepatosomatic index (HSI) showed a decreasing trend (P>0.05) with increasing dietary SL. The fatty acid composition of the hepatopancreas is largely due to the dietary composition. Higher dietary SL yielded a higher percentage of ovarian polyunsaturated and highly unsaturated fatty acids (P<0.05). Furthermore, the higher content of linoleic acid and alpha linolenic acid in diets may be more important for ovarian development in C. quadricarinatus, as these originate from the hepatopancreas. Our results suggest that dietary SL has a positive effect on vitellogenesis in C. quadricarinatus broodstock.2. Effects of dietary soybean lecithin on tissue ultrastructure, fatty acid-binding proteins and vitellogenin mRNA expressions in the female Cherax quadricarinatus.Did dietary SL promote vitellogenesis of C. quadricarinatus by promoting yolk nutrients accumulation? For the hypothesis, hepatopancreas and ovary ultrastructure, fatty acid-binding proteins (FABP) and vitellogenin (Vg) mRNA expressions of female redclaw crayfish, Cherax quadricarinatus, was investigated. With increasing percentages of dietary SL, the hepatopancreatic lipid droplets numbers and sizes increased and the structure and texture was more complete and distributed. The ovarian lipid droplets and yolk granules numbers and sizes increased. When dietary SL≥2%, shrimp hepatopancreatic and ovarian FABP mRNA expression increased with increasing dietary SL level (P<0.05). There were not significantly different between Diet1and Diet2for shrimp hepatopancreatic and ovarian FABP mRNA expressions (P>0.05), but they were lower than that of other treatments (P<0.05). The hepatopancreatic Vg mRNA expression was the highest in crayfish that received Diet3. Our results suggest that dietary SL can enhance ovarian nutrient accumulation in C. quadricarinatus broodstock.3. Effects of dietary soybean lecithin on lipid metabolic and physiological and biochemical index in the female Cherax quadricarinatus.In order to further understand physiological and biochemical mechanism of dietary lecithin affecting vitellogenesis and way of nutrients accumulation of Cherax quadricarinatus, we evaluate the effects of lipid metabolism and hepatopancreatic antioxidant capacity of shrimp fed diets containing increasing levels of SL(0%(Diet1),1%(Diet2),2%(Diet3),4%(Diet4)和6%(Diet5)). The hepatopancreatic lipid metabolism enzyme and hemolymph supernatant index were analyzed. Hepatopancreatic lipoprotein lipase (LPL) of shrimp in Diet5was higher than other four treatments(P<0.05). LPL in Diet4was not significantly different with that in Diet land Diet3(P>0.05), but it was higher than diet2(P<0.05). Shrimp hepatopancreatic hepatic lipase (HL) in diet1was not significantly different with Diet2(P>0.05), but it was higher than other three treatments(P<0.05). Total lipase was highest in diet5, following diet3and diet4, the lowest was diet1and diet2. When fed4%SL feed, shrimp hemolymph supernatant triglyceride was not significantly different to diet5(P>0.05), but significantly higher than other treatments (P<0.05). High density lipoprotein cholesterol in diet5was not significantly different to diet3and diet4(P>0.05), but significantly higher than diet1and diet2(P<0.05). When fed6%SL feed, shrimp hemolymph supernatant free fatty acid was not significantly different to diet4(P>0.05), but significantly higher than other treatments (P<0.05). Shrimp hepatopancreatic alanine aminotransaminase (ALT) in diet1and diet5were significantly lower than that in diet2and diet4(P<0.05). Aspartate aminotransaminase (AST) in diet5was significantly higher than other four treatments (P<0.05). Our results suggest that high dietary SL has a positive effect on hepatopancreatic and hemolymph supernatant lipid metabolism. When fed high SL feed, shrimp was still in steady-state of hepatopancreatic antioxidant system.Over all above results, dietary SL has a positive effect on vitellogenesis in C. quadricarinatus broodstock, which is accomplished by promoting rapid accumulation of nutrients and metabolism and absorption of lipid during vitellogenesis period. We suggest that at least2%SL should be supplemented in broodstock diets to enhance ovarian nutrient accumulation and ovary development during aquaculture.4. Molecular cloning, characterization expression and localization analysis of MPF in the red claw crayfish (Cherax quadricarinatus).Based on the results above, we further investigated the maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocytes, to try to understand the molecular nutrition mechanism. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized.4.1Characterization of Cdc2kinase in the red claw crayfish (Cherax quadricarinatus):Evidence for its role in regulating oogenesis.Cdc2kinase is a catalytic subunit of MPF, a central factor for inducing the meiotic maturation of oocytes. In this study, a complete cDNA sequence of Cdc2kinase was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. The Cdc2cDNA (1769bp) encodes for a299amino acids protein with a calculated molecular weight of34.7kDa. Quantitative real-time PCR demonstrated that Cdc2mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. With increasing dietary SL, shrimp ovarian Cdc2mRNA expression decreased firstly and then increased again. The expression was not significantly different among diet2, diet 3and diet4(P>0.05), but significantly lower than diet1and diet5(P<0.05). Immunohistochemistry analysis revealed that the Cdc2protein relocated from the cytoplasm to the nucleus during oogenesis.4.2Characterization of CyclinB in the red claw crayfish (Cherax quadricarinatus):Evidence for its role in regulating oogenesis.CyclinB is a regulatory subunit of MPF. The CyclinB cDNA (1779bp) of Cherax quadricarinatus encodes for a401amino acids protein with a calculated molecular weight and isoelectric point of34.7kDa and8.84. Quantitative real-time PCR demonstrated that CyclinB mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. With increasing dietary SL, shrimp ovarian CyclinB mRNA expression decreased firstly and then increased again. The expression was not significantly different among diet2, diet3and diet4(P>0.05), but significantly lower than diet1and diet5(P<0.05). Immunohistochemistry analysis revealed that the CyclinB protein also relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that MPF may play an important role in the gametogenesis and gonad development in C. quadricarinatus. And dietary SL influenced shrimp ovarian MPF mRNA expression.