Regulation Of Vitamin D₃ On Growth,Molting And Immunity Of Chinese Mitten Crabs （Eriocheir Sinensis）

As a steroid derivative,vitamin D₃（Vit-D₃）has a profound impact on the metabolism of the body.At present,studies on vertebrates focus on the effects of calcium and phosphorus homeostasis,bone mineralization,muscle function,lipid metabolism and immunity.However,studies on vit-D₃ invertebrates are mainly focused on the demand of economic crustaceans.Some of these studies have a long history and incomplete index evaluation,so that causing the optimal demand varies greatly,insufficient to support a systematic study of the physiological function of vit-D₃.As a symbolic biological phenomenon of crustaceans,molting not only runs through the whole growth and development cycle but also is a complex physiological regulation process similar to bone mineralization in vertebrates.However,the Chinese mitten crab（Eriocheir sinensis）,as one of the main aquatic economic crustaceans in China,has the phenomenon of failure in molting,small size and bacterial disease after molting,which not only affects the survival and growth of individual,but also directly affects the improvement of aquaculture output.Therefore,the key problems in E.sinensis industry at present are to ensure the normal molting of crabs by means of intensified nutrition and to cultivate disease-resistant seed.In order to ensure the normal growth and molting of E.sinensis in subsequent breeding,so as to adapt to diversified breeding environment conditions and improve the yield.Therefore,in this thesis,vit-D₃ was selected as the research object to explore the effects of vit-D₃ on growth,molting and immunity of crab at different growth stages,as well as physiological functions of crab under different nutritional conditions and its application strategies through the research methods of nutrition,molecular biology,immunology and transcriptomics.The main results and conclusions are as follows:1.Influences of dietary vit-D₃ on physiological function of E.sinensis larvaeTo investigate the effects of vit-D₃ on growth performance,antioxidant capacity,immunity and molting of larval Chinese mitten crab E.sinensis.A total of 6,000larvae（7.52±0.10 mg）were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary vit-D₃（0,3000,6000,9000,12000 and 36000IU/kg）respectively for 23 days.The highest survival and molting frequency were found in crabs fed 6000 IU/kg vit-D₃.Weight gain,specific growth rate,and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg vit-D₃ compared to the control.Broken-line analysis of molting frequency,weight gain and specific growth rate against dietary vit-D₃ levels indicates that the optimal vit-D₃ requirement for larval crabs is 4825–5918 IU/kg.The highest whole-body vit-D₃content occurred in the 12000 IU/kg vit-D₃ group,and the 25-dihydroxy vit-D₃content decreased with the increase of dietary vit-D₃.The malonaldehyde content was lower than the control.Moreover,the superoxide dismutase activity,glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg vit-D₃ were significantly higher than in control.Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress,and the relative m RNA expressions indicate vitamin D receptor（VDR）is the important regulatory element in molting and innate immunity.The molting-related gene expressions showed that the response of crab to salinity was self-protective.In conclusion,appropriate vit-D₃ can improve the growth performance,ensure normal molting activity,enhance innate immune and antioxidant capacity to cope with salinity stress,and thus increase survival rate of E.sinensis larvae.2.Impact of vit-D₃ metabolite and VDR on growth,molting and immunity of juvenile E.sinensisVitamin D₃（vit-D₃）,as an indispensable and fat-soluble nutrient,is associated with skeletal mineralization and health in mammals.However,such associations have not been well studied in economically important crustaceans.Six levels of vit-D₃ with isonitrogenous and isolipidic diets were used to feed E.sinensis.The range of optimal vit-D₃ requirements is 5685.43–10,000 IU/kg based on growth.The crabs fed 9000IU/kg vit-D₃ showed the best growth performance.This vit-D₃ dose significantly increased antioxidant capacity in the hepatopancreas and intestine and was optimal for molting and innate immunity via quantitative polymerase chain reaction analysis.Transcriptomics analyses indicate that vit-D₃ could alter protein processing in the endoplasmic reticulum,steroid biosynthesis,and antigen processing and presentation.As shown by the enzyme-linked immunosorbent assay,vit-D₃ could improve vitamin D receptor,retinoic acid receptor,and C-type lectins concentrations.The1α,25-dihydroxy vit-D₃ content in serum was significantly higher in 3000–9000 IU/kg vit-D₃.The result of si RNA interference showed that the expression level of VDR gene was highest in hepatopancreas.The inhibition efficiency of si RNA-74 and si RNA-200 on VDR gene reached 76.62%and 65.49%,respectively.The optimal concentration and frequency of injection were 2μg/g body weights,per 36 hours.RNAi results showed that si RNA group significantly inhibited VDR gene expression,Calcitriol group significantly increased VDR gene expression,si RNA+Calcitriol group could up-regulate VDR gene level,but still lower than NC group（control group）.Adequate dietary vit-D₃ can promote the growth,molting,antioxidant capacity and innate immunity of juvenile E.sinensis through 1α,25-dihydroxy vit-D₃and vitamin D receptor,while excessive dietary vit-D₃ will have a negative influence.3.Effect of different levels of calcium and vit-D₃ on growth,calcium-phosphate deposition and innate immunity of juvenile E.sinensis Considering that dietary vit-D₃ level significantly affected ash content,molting frequency and molting related gene expression of crab in the study of dietary requirement level.In addition,dietary calcium levels have been shown to affect the absorption of calcium and phosphorus by vit-D₃ in vertebrate studies,to evaluate the synergistic effect of vit-D₃ and Ca in molting and immunity in Chinese mitten crab（E.sinensis）,the crabs（3.20±0.01 g）were fed six diets formulated with two Ca levels（0%and 1.5%,abbreviated D and N）and three vit-D₃ levels（0,9000 and 27000 IU/kg, abbreviated LVD,MVD and HVD）level for 56 days.Crabs fed 1.5%Ca and 9000IU/kg Vit-D₃（N+MVD）had the highest weight gain,specific growth rate,molting frequency,hematic-1,25-dihydroxy vit-D₃,hematic-calcium,hepatic-phosphorus,glutathione peroxidase,total antioxidant capacity,and the lowest malondialdehyde and feed conversion ratio among groups.On the contrary,crabs fed diets without Ca had low weight gain,molting frequency,trypsin activity,hepatic-calcium, sarcous-phosphorus,antioxidant capacity,and the expression of molting-related and immune-related genes.Compared to crab fed the N+HVD（1.5%Ca+27000 IU/kg vit-D₃）diet,those fed the D+HVD（0%Ca+27000 IU/kg vit-D₃）diet had significantly lower weight gain,specific growth rate,trypsin activity,and gene expressions of crustacean hyperglycemic hormone,chitinases,ecdysone receptor,retinoid X receptor,toll-like receptor 2,lysozyme,anti-lipopolysaccharide factor,peritrophin-44-like protein and peritrophin.This study demonstrates that adequate dietary calcium and vit-D₃ can enhance growth,molting activity,antioxidant capacity and innate immunity in E.sinensis.But when calcium is deficient in the diet, excessive vit-D₃ supplementation will make the negative effects.4.Impact of different levels of lipid and vit-D₃ on antioxidant capacity,lipid metabolism and innate immunity of juvenile E.sinensisAs a fat-soluble vitamin,vitamin D₃ not only relies on fat to play its biological function,but also affects the body’s fat metabolism.To evaluate the synergistic effects of dietary lipid level and vit-D₃ level on growth,lipid metabolism and immunity of juvenile E.sinensis,the crabs（5.83±0.01 g）were fed six diets formulated with two lipid levels（1.5%and 7.5%,abbreviated LL and NL）and three vit-D₃ levels（0,9000and 27000 IU/kg,abbreviated LVD,MVD and HVD）level for 56 days.The synergistic effect of dietary lipid level and vit-D₃ level had no significant effect on growth performance,but significantly affected ash content,total protein,hepatopancreas lipid content,serum 1α,25-hydroxy vit-D₃ content,hepatopancreas lipolysis and synthesis genes.Crabs fed 7.5%Ca and 9000 IU/kg Vit-D₃ had the highest hepatopancreas index,serum 1α,25-hydroxy vit-D₃ content,antibacterial ability,expression of molting-related and immune-related genes,and expression of hepatopancreas lipid synthesis genes,but the expression of lipolysis genes became lower.On the contrary,crabs fed diet with lower lipid level（1.5%）had low growth performance,serum 1α,25-hydroxy vit-D₃,lipid synthesis genes m RNA level,antibacterial ability and immune-related genes expression.When at 1.5%lipid level,excessive or lack of vit-D₃ supplementation will lead to the obstruction of ash and protein deposition,which will hinder the growth and molting,aggravate the reduction of antioxidant capacity,hinder the antimicrobial peptides genes expression and then affect innate immunity,and lead to lipid metabolism disturbance,resulting in abnormal lipid accumulation in juvenile crabs,bringing the risk of oxidative stress.This study demonstrates that adequate dietary lipid and vit-D₃ can enhance antioxidant capacity,lipid metabolism and innate immunity in E.sinensis.While low lipid level diet will not only cause growth retardation,but also reduce antioxidant capacity,innate immunity and give rise to lipid metabolism disorders.