Studies On Vitellogenesis Of Cherax Quadricarinatus

Redclaw crayfish(Cherax quadricarinatus)subordinated to Crustacea, Decapoda,Pleocyemata,Astacidea,Parastacoidea,Parastacidae,Cherax.It is a robust species with broad geographical potential,simple life cycle,fast growing,good adaptability,simple foods,approving freshwater raising and many other merits for commercial production.Redclaw has been introduced for aquaculture to many countries and area until the end of 80's.It was fetched in our country in 1992,and had some successful breed reports,but still hadn't come into large scale cultivation,for some key problems have not been solved such as mature synchronization rate,low rate of spawning and hatching,livability.Vitellogenesis is the necessary condition for the mature of oocyte in crustaceans,a crucial period in reproduction for female crustaceans,connected with the utilizing of nutrition in embryo.So,the vitellogenesis is very important for redclaw's reproduction and it would influence the hatching rate and the fetation.This study based on previous studies,methods of histlolgy,electron microscope were used to describe the characteristic of vitellogenesis and different organelles in the process,biochemical,enzymology and molecule methods were used to detect the biochemical and physiology index.The data obtained would provide valuable information to a better understanding on vitellogenesis of crustaceans,and it is helpful to redclaw's commercial breeding.1.Morphology of Cherax quadricarinatus during vitellogenesis1.1 Study on the microstructure and ultrastructure of ovary during vitellogenesis of Cherax quadricarinatusThe vitellogenesis of the oocytes in Cherax quadricarinatus was examined by electron microscopy.The result indicate a dual origin of the york,but the exogenous yolk was more than endogenous yolk.All kinds of organelles were involed in vitellogenesis,organelles which have strong anabolic effect were lack of amount,they were concerned with the formation of endogenous yolk and precursors of york granule,such as ribosome,endoplasmic reticulum and golgi apparatus.But organelles involed in exogenous yolk formation were abundance and developed, lysosome engulfe and digest inclusion to form yolk granules with endoplasmic reticulum and other organelles;aside from offering energy for other organelles,lots of mitochondrion also participate in the formation of yolk granules.Though the endogenous yolk formation take part in the primary vitellogenesis and active vitellogenesis,the exogenous yolk formation played the most important role in the whole process of vitellogenesis,especially in active vitellogenesis and later vitellogenesis.1.2 Study on the microstructure and ultrastructure of hepatopancreas during vitellogenesis of Cherax quadricarinatusThe histology and ultrastructure of the hepatopancreas during the vitellogenesis of Cherax quadricarinatus were studied with light and electron microscopes.The result indicated that the epithelium cells of hepatopancreatic tubules are classified into R-cell,F-cell,B-cell and E-cell based on structure and function.During the process of vitellogenesis,there are big amount of R-cells,and the proportion of R2-cells is increasing;the exogenous nutrientsubstance absorbed by R-cells is synthesized and packed afresh by organelles such as endoplasmic reticulums,mitochondrias and ribosomes to form vitellogenin granules and lipid droplets.Rough endoplasmic reticulums and mitochondrias are rich in F-cells which shows strong function on synthesizing zymogen granules and their amount is droping off during the vitellogeneis.B-cells have a large digestive vacuole,they have functions of absorption,digestion and secretion.E-cells are located in the edge of hepatopancreatic tubules and divide by mitosis to differentiate into other type of cells.The formation of vitellogenin granules and lipid droplets was described detailedly and compared with that in ovary,the function of cytoplasmic organelles during vitellogenesis was discussed too.Based on the histology and ultrastructure results of hepatopancreas and ovary and the data of QRT-PCR,we consider that the exogenous yolk formation played the most important role in the whole process vitellogenesis of Cherax quadricarinatus.2.Study on biochemical composition and the digestive enzyme of Cherax quadricarinatus during vitellogenesis2.1 Study on biochemical composition of Cherax quadricarinatus during vitellogenesisThe compositions and content of water,soluble protein and amino acids,lipid and fatty acid were measured and analyzed by biochemical methods.The results indicates that the content of water,soluble protein and lipid in hepatopancreas trends down during vitellogenesis;and in ovary,the content of soluble protein has an obvious ascend in stage 8;the lipid content continual rising and the water content kept on deceasing.The change of those biochemical content incarnates the characteristic how material utilize and transfer during vitellogenesis.During vitellogenesis,the content of total amino acids fluctuated and had a slightly downtrend in hepatopancreas;and in ovary,the content of total amino acids had a uptrend,and significant differences exist in different stasges.There were 17 amino acids,which contain 9 EAA and 8 NEAA in hepatopancreas and ovary during vitellogenesis.Leu and Arg content were the highest both in hepatopancreas and ovary, and in ovary Lys and Val also has a high content.The change of amino acids composition was connected closely with vitellogenesis.The SFA content in hepatopancreas during vitellogenesis decreased significantly, from 35%in stage 1 to 29%in stage 8.The content of MUFA changed from 30%in stage 1 to 40%in stage 8;the PUFA fluctuated but changed little.The quantitatively most important fatty acids in hepatopanxreas were the saturates(SFA):C16:0,the monounsaturates(MUFA):C18:1n9c,the polyunsaturates(PUFA):C18:2n6c.The SFA in ovary decreased significantly too,from 48%in stage 1 to 23%in stage 8;the content of MUFA in ovary increased remarkably changed from 33%in stage 1 to 49% in stage 8;the content of PUFA in ovary also had a significant uptrend which differ from that in hepatopancreas.The quantitatively most important fatty acids in ovary were the saturates(SFA):C 16:0,the monounsaturates(MUFA):C 16:1 and C 18:1 n9c, the polyunsaturates(PUFA):CI 8:2n6c and C20:5n3.2.2 Study on digestive enzyme activities of Cherax quadricarinatus during viteilogenesisBiochemical methods were employed to determine several digestive enzyme in hepatopancreas and ovary during vitellogenesis of Cherax quadricarinatus,including: lipase,HL,acid phosphatase,alkaline phosphatase,tryptase,cellulose and amylase. The results indicated that during vitellogenesis the activity of lipase in hepatopancreas elevated from stage 1 to stage 6,decreased rapidly in stage 7,and rised again in stage 8;the activity of lipase in ovary stayed on a low level and fluctuated slightly.The activity change of HL showed an enantiomorphous trend which was opposite from the trend of change that lipase displayed.In hepatopancreas,the activity of acid phosphatase was highest in stage 1,2,decreased at stage 3;and displayed a low level in ovary.The activity of alkaline phosphatase was very close both in hepatopancreas and ovary,except a significant ascend at stage 5 in hepatopancreas.In hepatopancreas, the activity of tryptase fluctuated obviously,and in ovary,the the activity of tryptase was in lower level.The activity of amylase in hepatopancreas had a uptrend with a little fluctuation;and in ovary the activity was steady from stage 1 to stage 5,declined from stage 6 to stage 8.The activity of cellulose in hepatopancreas kept steady during vitellogenesis,in ovary the activity of cellulose was highest at stage 2 and decreased at the rest stages.The hepatopancreas is the most important digest gland in crustaceans,the impostant place for exogenous vitellogenesis;the ovary is the important place for endogenous vitellogenesis and also the destination of exogenous yolk,thus the change of activity of digestive enzyme can be good response to the digest physiological change,material metabolizability and transfer during vitellogenesis.3.Differential expression of vitellogenin(Vg)and farnesoic acid O-methyltransferase(FaMet)of Cherax quadricarinatus during vitellogenesis3.1 Expression of vitellogenin(Vg)of Cherax quadricarinatus during vitellogenesisThe expression of vitellogenin(Vg)of Cherax quadricarinatus during vitellogenesis was detected by QRT-PCR methods.The results indicated that during vitellogenesis,the expression of Vg in hepatopancreas showed itself a significant uptrend,the relative quantity of the Vg mRNA at stage 1 and stage 2 was low,and from stage 3 to stage 8 it rose rapidly,except little descend at stage 5 and stage 7.In ovary,the Vg gene expression quantity was very low.Viewed from the whole process of vitellogenesis,Vg is mainly synthesize in hepatopancreas and the ovary hardly had contribution to vitellogenesis.3.2 Expression of farnesoic acid O-methyltransferase(FaMet)of Cherax quadricarinatus during vitellogenesisFaMet is the enzyme responsible for final step in the conversion of FA to MF, and studies on MF indicate that it is involved in many important biological functions in crustaceans,including metamorphosis,reproduction and moulting.QRT-PCR methods was used to detect the expression of FaMet both in hepatopancreas and ovary. The result indicated that the expression of FaMet fluctuated during vitellogenesis,the quantity was low at stage 1,from stage 2 to stage 4 it had a significant uptrend,during stage 5 and stage 7 it had a little descend and rose again at stage 8.In hepatopancreas, the FaMet mRNA levels werer lower than that in ovary,and change lessly during vitellogenesis.The presence of FaMet in target tissues is for the final conversion to MF or the FaMet RNA encodes another relational protein that needs further study.