Identification Of Intestinal Microbiotas In Grass Carp With Different Growth And Functional Research Of Short-chain Fatty Acid Receptor Gene

Grass carp(Ctenopharyngodon idella)is an important freshwater fish in China.Growth traits are important aims for grass carp breeding program.The intestinal microbiotas and its metabolites,short-chain fatty acids(SCFAs),are important factors in regulating growth of grass carp.Our research characterized the digesta and mucosal microbiome of the different regional intestines in grass carp.The association between different body-mass and grass carp intestinal microbiota was compared in the study.The regulatory mechanism of SCFAs on the appetite of grass carp was investigated in the study.The structural and functional studies of grass carp GPR41 and GPR43 genes were researched in the study.The SNPs of GPR41 gene associated with grass carp growth traits were developed in the study.The main results of the study were as follows:1.The digesta and mucosal microbiome were characterized in the anterior,middle,and posterior intestine of the grass carp,using 16 S rRNA next-generation sequencing.As results,Proteobacteria,Firmicutes and Bacteroides were the dominant phyla in the intestine of grass carp.The microbial communities of the middle intestine exhibited higher alpha diversity indices compared with the anterior and posterior intestine.The anterior and middle intestines were similar in microbiomes,but were significantly different to the posterior intestine based on the NMDS,PERMANOVA and UPGMA analysis(P < 0.05).We observed a significantly increased abundance of cellulose-degrading microbiomes in the posterior intestine by linear discriminant analysis(LDA)coupled with effect size(LEf Se)analysis(P < 0.05).Our results showed that the microbiomes of the posterior intestine were distinct from the anterior and middle intestinal microbiotas in grass carp.2.The association between different body-mass and intestinal microbiome of grass carp was compared by next-generation sequencing of the 16 S rRNA gene.The results showed that alpha diversity indices of the low-weight-gain(LWG)groups were higher than that of the high-weight-gain(HWG)groups.HWG groups possessed the decreased ratio of Bacteroidetes to Firmicutes compared with that in the LWG groups.PCo A and ANOSIM revealed that there were significant differences between the HWG and LWG groups(P < 0.05).Furthermore,LEf Se analysis showed that the intestinal microbiota that metabolizing SCFAs were significantly abundant in the HWG groups(P < 0.05).p MENs revealed that more complex microbial interactions were found within HWG group,as evidenced by lower GD,higher avg CC and avg K.Our results demonstrated that there appeared to be a tight correlation between the intestinal microbiota and body-mass in grass carp.Additionally,we found SCFAs were the key factors affect the body-bass of grass carp.3.The appetite genes of intestine and brain,as well as intestinal enzyme activities,was investigated in grass carp following SCFAs injection.As for intestine,the results showed that the relative expression of PYY gene was significantly decreased at 4h post-injection,the relative expression of NPY gene was significantly increased at 4h post-injection,the relative expression of CCK gene was significantly decreased at 4-12 h post-injection,and the relative expression of GPR41 and GPR43 genes appeared to be increased at 4-12 h post-injection(P < 0.05).With regard to brain tissues,the relative expression of NPY gene was significantly increased at 8-12 h,and the expression of CCK gene was significantly decreased at 4-8h post-injection(P < 0.05).The intestinal amylase,lipase and protease activities performed significantly increased following SCFAs injection(P < 0.05).The results indicated that injection of SCFAs affected the transcriptional levels of appetite regulatory factors in the intestine and brain tissue,while increasing the intestinal digestive enzymes activity of grass carp.4.The gene structure,tissue expression,and regulatory mechanisms of grass carp GPR41 and GPR43 were researched in the study.The full-length cDNA of grass carp GPR41 and GPR43 were 1609 bp and 2491 bp,respectively.The tissue distribution profile showed that GPR41 and GPR43 transcriptional levels were significantly high in the intestine and spleen(P < 0.05).The functional assays demonstrated that Gαi/o-AC-c AMP pathway was implicated in SCFA-mediated GRP41 activation.Both Gαi/o-AC-c AMP pathway and PLC pathway were involved in SCFA-mediated GRP43 activation.Calcium assay showed that SCFAs increased calcium mobilization by activating GPR41 in a Gαi/o-dependent manner,however,GPR43-mediated calcium mobilization was not dependent on Gαi/o.Our findings suggested that the GPR41 and GPR43 played important mediating roles in the process of SCFAs physiological functions in grass carp.5.The single nucleotide polymorphisms(SNPs)of GPR41 gene associated with grass carp growth traits were developed by direct sequencing in the study.As a result,seven loci were not significantly deviated from Hardy-Weinberg equilibrium(P > 0.05).Haplotype analysis found a total of five haplotypes,with HAP1(52.5%)according for the highest percentage.Grass carp body mass and length were significantly correlated with the 1422 C > T locus genotype,and its heterozygous mutant(CT)had significantly higher body mass and length than the wild type(CC)by general linear model analysis(P < 0.05).The results demonstrated that the 1422 C>T locus of GPR41 gene was significantly associated with growth traits,and it can be used as candidate assistant markers for the growth improvement of grass carp.