| In this study,temperature was taken as a single factor variable,and three modes of high temperature stress,low temperature stress and temperature change were set to explore the effects of temperature change on antioxidant system,non-specific immune system,expression of immunerelated genes,intestinal microorganisms and metabolome of Eriocheir sinensis.The purpose of this study was to explore the adaptive mechanism of Eriocheir sinensis to temperature change from the aspects of enzymology,gene expression,intestinal microbiome and omics,so as to provide a theoretical basis for guiding the breeding and production of Chinese mitten crab.The main research results are as follows:(1)High temperature stress experiment: With 24℃ as the control and the water temperature rising from 24℃ to 32℃,the effects of high temperature stress on physiology and biochemistry,antioxidant system,non-specific immune system,intestinal microorganisms and hepatopancreas metabolome of Eriocheir sinensis were studied.The results showed as follows: 1)Under high temperature stress,Glu content in hemolymph of Eriocheir sinensis was significantly higher than that in control group during 24-96 h(P<0.05);The level of TC in hemolymph was significantly higher than that in control group at 12 h to 7 d(P<0.05).There was no significant change in TP content(P>0.05).LDH activity in hemolymph increased first and then decreased,and reached its peak at 12 h.2)Under high temperature stress,the activities of antioxidant enzymes CAT,GSH and SOD in hepatopancreas of Eriocheir sinensis increased first and then decreased(P<0.05),and CAT and GSH peaked at 24 h.CAT,GSH and SOD were significantly higher than those in control group at 12-24 h,6-72 h and 6-96 h,respectively(P<0.05).MDA content also increased firstly and then decreased with stress time(P<0.05).T-AOC was significantly lower than that of control group in48 h to 7d(P<0.05).3)The activities of immune-related enzymes AKP,ACP,AST and ALT were firstly increased and then decreased(P<0.05).4)In hepatopancreas,the expression levels of immune-related genes HSP70,HSP90,pro PO and Relish are significantly increased under high temperature stress,and the expression levels of HSP70,HSP90 and Relish are significantly higher than that of control group(P<0.05).5)High temperature stress significantly affected the intestinal microbial composition of Eriocheir sinensis,and the abundance of beneficial bacteria Proteobacteria,Campilobacterota and Candidatus＿Hepatoplasma significantly decreased.The harmful bacteria Actinobacteriota,Rhodococcus and Morganella were significantly increased(P<0.05).6)After high temperature stress,22 differential metabolites(SDMs)were significantly up-regulated and 63 SDMs were significantly down-regulated(P<0.05),including amino acids and their metabolites,fatty acids and heterocyclic compounds.These SDMs were enriched and annotated in 34 different metabolic pathways.(2)Low temperature stress experiment: With 24℃ as the control,the water temperature decreased from 24℃ to 12℃ to study the effects of low temperature stress on physiology and biochemistry,antioxidant system,non-specific immune system,intestinal microorganisms and hepatopanacolic metabolome of Eriocheir sinensis.The results showed as follows: 1)Under low temperature stress,Glu content in hemolymph of Eriocheir sinensis was significantly decreased at12 h(P<0.05),but was significantly increased at 24 h,72 h and 96 h(P<0.05).No significant changes were found in TP level(P>0.05).Low temperature stress significantly increased TC content in hemolymph at 6 h and 96 h(P<0.05).LDH activity initially decreased and then increased after low temperature stress(P<0.05).2)Under low temperature stress,the levels of antioxidant enzymes CAT,GSH and SOD were firstly increased and then decreased,and CAT,GSH and SOD were significantly higher than those of control group at 24-48 h,12-72 h and 12-96 h,respectively(P<0.05).After 12 h stress,T-AOC in low temperature group was significantly decreased(P<0.05).3)The activities of immune-related enzymes AKP,ACP,AST and ALT were significantly increased in the late stage of low temperature stress(P<0.05).4)The expressions of immune-related genes HSP70,HSP90 and pro PO were significantly higher than those of control group during 48-96 h after low temperature stress(P<0.05).The expression of gene on Relish is significantly increased at 12 h,24 h,and 72 h(P<0.05).5)The low temperature stress on eriocheir sinensis gut microbe structure have a significant impact,in the level,the low temperature stress significantly increased the Dechloromonas,Mariniflum,and unclassifed-f-Comamonadaceae relative abundance,The relative abundance of Chitinibacter and unclassified-c-Alphaproteobacteria was significantly decreased(P<0.05).6)After low temperature stress,9 SDMs were significantly up-regulated and 8 SDMs were significantly down-regulated(P<0.05),mainly including amino acids and their metabolites,benzene and its substituted derivatives,fatty acids,alkaloids,hormones and related compounds.These SDMs were enriched and annotated in 10 different metabolic pathways.(3)variable-temperature culture experiment: Ct(24±0℃)in control group and Vt-1(24±1.5℃),Vt-2(24±3.0℃),Vt-3(24±4.5℃)and Vt-4(24±6.0℃)in variable-temperature group were set respectively.The temperature in variable-temperature group changed periodically every day,and the culture cycle was 51 d.The results showed as follows: 1)the weight gain rate of juvenile crabs in Vt-3 group was significantly higher than that in control group and other variable temperature groups(P<0.05).2)Glu content in hemolymph of crab in Vt-1 group was significantly higher than that in Vt-2 group(P<0.05);The TP content in crab hemolymph in groups Vt-1 and Vt-2 was significantly higher than that in control group and Vt-4 group(P<0.05),but there was no significant difference among the other three groups(P>0.05).There were no significant differences in TC content and LDH activity in hemolymph between the control group and the other 4experimental groups(P>0.05).3)The activities of CAT and SOD in crab hepatopancreas in Vt-4group were significantly higher than those in the other four groups(P<0.05);The content of T-AOC in hepatopancreas of crabs in Vt-2 group was significantly lower than that in the other four groups(P<0.05),and that in Ct and Vt-1 groups was significantly lower than that in Vt-3 and Vt-4 groups(P<0.05).MDA content and GSH activity in crab hepatopancreas were not significantly different between control group and the other four thermotropic experimental groups(P>0.05).4)The intestinal microbial composition of mitten crab was significantly affected by different temperature change conditions.At the gate level,the relative abundance of Planctomycetota in temperature change group was significantly higher than that in control group(P<0.05).At the genus level,the relative abundance of Mycobacterium,Hyphomicrobium,Candidatus-Ovatusbacter and Clostridium-sensu-stricto-7 were significantly increased in the vari-temperature group(P<0.05).5)The metabolites of hepatopanpancreas of Eriocheir sinensis were significantly affected by different temperature changes.Compared with Ct group,15 SDMs in Vt group were significantly upregulated(P<0.05)and 16 SDMs were significantly down-regulated(P<0.05),mainly including amino acids and their metabolites,glycerol phospholipids,fatty acids,heterocyclic compounds,organic acids and their derivatives.These SDMs enriched annotations were found in 34 different metabolic pathways. |
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