| Chinese mitten crab(Eriocheir sinensis)is an important economic crustacean in native China,and its growth is closely related to molting.Considerable differences in weight gain rates(WGRs)after molting is a puzzling phenomenon exhibited among E.sinensis individuals even under the same environmental conditions,which seriously affect the production and the economic benefit of E.sinensis aquaculture.Therefore,this study focused on the biological problem of the difference in WGRs of E.sinensis after molting.Juvenile E.sinensis individuals were cultured in the same environment during one molting cycle to obtain crab individuals with different WGRs.The multi-omics techniques integrated with corresponding molecular function verification were adopted to explore the regulation mechanism of weight gain difference of E.sinensis during the molting cycle.The main results are presented as follows:1.A study on the mechanism of regulating the difference in weight gain of E.sinensis underlying the different molting stagesIt is unknown whether different molting stages have different effects on the weight gain of E.sinensis after molting.In this chapter,taking into account the different molting stages,the regulation of weight gain in E.sinensis during the whole molting cycle(InM,PrM and PoM)was preliminary discussed.Juvenile E.sinensis individuals were cultured in the same environment during one molting cycle.After molting,the E.sinensis individuals were classified into the high-and low-WGR groups on the basis of WGRs.Hemolymph samples were collected at the intermolt(InM),premolt(PrM)and postmolt(PoM)stages of the molting cycle for biochemical index determination,metabolomic and transcriptomic analyses.The results of biochemical indexes in plasma showed that:the content of glucose in the high-WGR group increased significantly at the PrM stage and was higher than that in the low-WGR group(P<0.05);the concentration of 5-HT in the high-WGR group was significantly higher than that in the low-WGR group at the InM stage(P<0.05).Differential metabolites(DMs)and differentially expressed genes(DEGs)at the InM,PrM,and PoM stages were identified and mapped to Kyoto Encyclopedia of Genes and Genomes pathways.Integrated metabolomic and transcriptomic analyses revealed key DMs,DEGs and significantly varied metabolic pathways between different WGR groups at the three molting stages.A hypothesis for underlying mechanism in controlling E.sinensis weight gain during the molting cycle was proposed preliminarily.At the InM stage,tryptophan/phenylalanine/lysine metabolism,oxidative phosphorylation,tricarboxylic acid cycle,endoplasmic reticulum protein processing,ribosome and glutathione metabolism were enriched in the highWGR group;In the PrM stage,tryptophan/phenylalanine metabolism,synaptic vesicle cycle,oxidative phosphorylation and glutathione metabolism were enriched in the highWGR group;In the PoM stage,tryptophan/phenylalanine metabolism,fatty acid degradation and glutathione metabolism were enriched in the high-WGR group.Thus,the present results showed that the high-WGR group exhibited more active cellular protein biosynthesis,a robust neuroendocrine system,and higher antioxidant and detoxifying capabilities than the low-WGR group.These characteristics may promote weight gain during the molting cycle.This study provided novel insights into the molecular and metabolic mechanisms in controlling the growth of E.sinensis during the molting cycle.2.A further study on regulating weight gain differences of E.sinensis at the InM stageTo further analyze the molecular mechanism underlying the difference in weight gain rate of E.sinensis,two tissues,muscle and hepatopancreas,which closely related to growth were selected for gene expression profile analysis to explore functional candidate genes.In this chapter,juvenile E.sinensis individuals were cultured in the same environment during one whole molting cycle for the second time.The muscle and hepatopancreas tissues were sampled 15 days after the second molting(InM),and two groups of samples with significant difference in WGR(high-and low-WGR groups)were screened out for transcriptome sequencing analysis.The gene expression profiles between the different WGR groups were compared and analyzed.The results showed that:In muscle,up-regulated genes in the high-WGR group were mainly enriched in metabolic processes related to muscle development,while down-regulated genes were mainly enriched in N-glycation related processes,the DEGs included Actin1,myosin heavy chain(MYH),tubulin,early growth response protein 1(EGR1),growth arrestspecific protein 1(GAS1),activin receptor ⅡB(ActRⅡB)and related genes;in hepatopancreas,up-regulated expressed genes in the high WGR group were mainly enriched in metabolic pathways such as oxidative phosphorylation,while downregulated expressed genes were mainly enriched in fatty acid degradation,amino acid catabolic metabolism and other related processes;the DEGs included fatty acid-binding protein(FABP),fatty acid elongase protein(FAE),acyl-coenzyme A amino acid Nacyltransferase(ACOT1),ATP synthase(ATPase),cytochrome c oxidase(COX3),NADH dehydrogenase(NADH5)and related genes.The results of glucose content in plasma showed there was no significant difference between the different WGR groups(P>0.05),and the results of 5-HT concentration in plasma showed that of the high-WGR group was significantly higher than that of the low WGR group(P<0.05).Those results showed that E.sinensis individuals with high-WGR promote muscle growth by accelerating muscle protein synthesis at the InM stage and accumulate more energy by speeding up the lipid synthesis in hepatopancreas for the next ecdysis,while provide large amounts of energy for the muscle growth through the glucose oxidation decomposition,and thus to accomplish the regulation of rapid weight gain after molting.3.Study on molecular characteristic and function of growth candidate gene Es-ActRⅡBActivin receptor ⅡB(ActRⅡB)is a serine/threonine-kinase receptor binding with transforming growth factor-β(TGF-β)superfamily ligands to participate in the regulation of muscle growth and lipid metabolism in vertebrates.In this chapter,the E.sinensis ActRⅡB(Es-ActRⅡB)gene was used as a growth candidate gene,and the study on its molecular structure analysis and its function of participating in the weight gain during the molting cycle were carried out.(1)The cDNA sequence of Es-ActRⅡB was cloned by using rapid-amplification of cDNA ends,and obtained with a 4,916 bp full-length cDNA,including a 1,683 bp open reading frame that encoded 560 amino acids,which contains the characteristic domains of TGF-β type Ⅱ receptor superfamily.(2)The expression level of Es-ActRⅡB in 11 different tissues at four different molting stages(PoM,InM,PrM and E)was detected by using qRT-PCR.The mRNA expression of EsActRⅡB was the highest in hepatopancreas and the lowest in muscle at each molting stage,and reached the highest at the PrM stages(P<0.05).(3)Further dsRNA interference technology was used to knockdown Es-ActRⅡB gene expression,and after persistently interfering with a whole molting cycle,the growth characteristics of E.sinensis was evaluated,the amino acids and fatty acids composition in muscles and hepatopancreas were analyzed,and the expression levels of related genes were detected.The results showed that:the RNA interference(RNAi)group showed higher weight gain rate,higher specific growth rate,and lower hepatopancreas index compared with the control group(P<0.05).Meanwhile,the RNAi group displayed a significantly increased content of hydrolytic amino acid(TEAA,TNEAA and TAA)in both hepatopancreas and muscle.The RNAi group also displayed slightly higher contents of saturated fatty acid and monounsaturated fatty acid with no significantly difference(P>0.05);but significantly decreased levels of polyunsaturated fatty acid in hepatopancreas compared with the control group(P<0.05).After RNAi on Es-ActRⅡB,the mRNA expressions of five ActRⅡB signaling pathway genes showed that ActRⅠ and forkhead box O(FoxO)were downregulated in hepatopancreas and muscle,but no significance expression differences were found in small mother against decapentaplegic(SMAD)3,SMAD4 and mammalian target of rapamycin.The mRNA expressions of three lipid metabolism-related genes(carnitine palmitoyltransferase β(CPT1β),fatty acid synthase,and fatty acid elongation)were significantly downregulated in both hepatopancreas and muscle with the exception of CPT1β in muscles.These results indicate that ActRⅡB is a functionally conservative negative regulator in growth mass,and protein and lipid metabolism could be affected by inhibiting ActRⅡB signaling in E.sinensis.4.The gut microbiome composition of E.sinensis with different weight gain ratesIn order to explore the relationship between the composition of gut microbiome and the weight gain difference of E.sinensis during the molting cycle,16S rDNA sequencing was used to analyze the differences of composition,structure and function of gut microbiome in the high-and low-WGRs groups at the InM stages.The results showed that:The gut microbiome of E.sinensis were mainly composed of Firmicutes,Proteobacteria and Bacteroidetes.The richness of gut microbiome(Ace index)in the high-WGR group was significantly higher than that in the low-WGR group(P<0.05),but there was no significant difference in gut microbiome diversity(Shannon and Simpson index)between the different WGR groups(P>0.05).At the level of phylum classification,there was no significant difference in gut microbiome composition between the different WGR groups(P>0.05),but the specific phylum of Spirochaetota and Acidobacteriota with the lower abundance were detected only in the high-WGR group.At the level of genus classification,Flavobacterium,norank_f_Kineosporiaceae and C1500-29_marine_group in the high-WGR group were significantly higher than that in the low-WGR group(P<0.05),and 56 specific genus such as Bifidobacterium and Ruminococcus_torques_group were only found in the high-WGR group.Functional prediction analysis by using PICRUSt showed that the abundance of carbohydrate metabolism,amino acid metabolism,lipid metabolism and energy metabolism of gut microbiome in the high-WGR group were higher than those in the low-WGR group.The results indicated that differences in the gut microbiome composition between different weight gain rates of E.sinensis,and the specific beneficial bacteria in the gut microbiome might play an important role in the weight gain of E.sinensis during the molting cycle. |
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