Correlation Of Insulin-like Genes With Glycogen Content And Gonadal Development In Crassostrea Gigas

The insulin-like superfamily is an ancient gene family in animals,which is involved in the regulation of a variety of physiological processes,including reproduction,development,growth,carbohydrate and lipid metabolism.There are many insulin-like peptide genes(ILPs)have been found in shellfish,but research on their function is scant and it is unclear what specific biological processes these genes are involved in.In this study,we selected Crassostrea gigas as experimental animal to assay the possible involvement of oyster ILPs in the regulation of glycogen content and gonad development.Physiological condition and gene expression levels were assayed on oysters collected monthly from the oyster farm,multivariate statistical analysis was then applied to study the correlations between ILP expression and physiological conditions.RNAseq was conducted on the hepatopancreas of oysters injected with glucose to assay the transcriptomic response to the blood glucose increase.The results are listed as follows:Glycogen content variations during natural state of Crassostrea gigas and their correlations with the expression patterns of insulin-like genes(cgMIP123,cgMIP4,cgMILP7,cgILP).The blood glucose content,glycogen content,condition index and relative gene expression level were assayed on oysters cultured in a farm at Jiaonan,Shandong,while typical marine environmental factors(acidity,temperature,salinity,Chla)were collected from May 2020 to May 2021.Multivariate logistic regression analysis indicated high correlation between the spawning and variations of blood glucose content and glycogen content around pyloric process.A regression model was constructed to estimate the gonad discharging status by the two parameters.The concordance index was at 0.903,while theχ~2 value was at 9.06(p-value>0.05)for Hosmer-Lemeshow test,suggesting high goodness of fit.Application of the model to verification samples also showed high reliability.The multiple linear regression analysis showed correlations of growth or physiological values with the expression pattern of some genes:the condition index was correlated with cgMIP123,cgMIP4and cgMILP7(R~2:0.83,p-value:7.87e-04);cgMIP4 and cgILP were positively correlated with glycogen content;there is no correlation between cgMIP123 and glycogen content;the relative expression of cgMILP7 was negatively correlated with glycogen content of all the assayed tissues,this gene may be related to gonadal development and has partial correlation in blood glucose regulation.After intramuscular injection of glucose,the blood glucose level of Crassostrea gigas increased rapidly in a short period of time,and then decreased gradually in a fluctuating way,and returned to the initial level 2 hours later.Transcriptome analysis of hepatopancreas of oysters after injection of glucose stimulation showed that insulin-like genes were not concentrated in differential genes,which may indicate that there was no change in insulin-like genes at the transcriptional level.cgPPP1R3B and cgPCSK1 were selected as two differential genes from the differential gene set. cgPPP1R3b played an important role in the process of glycogen synthesis,and cgPCSK1 plays an important role in the processing of ILP polypeptides,indicating enhanced regulation of ILP at the post-translational level.The results of starvation experiment did not show significant differences in blood glucose and glycogen content,which may be related to the short experiment period and fewer biological replicates.