Changes Of Physiological And Internal Signals Of Chinese Mitten Crab(Eriocheir Sinensis) Under Pesticide Stress

Chinese mitten crab(Eriocheir sinensis),also known as the river crab,is an important economic species in freshwater aquaculture in China with delicious taste and high nutritional value.High-density and intensive aquaculture are beginning to occur in river crab farming,and,pyrethroid pesticides,avermectin,and trichlorfon are often used to clear ponds and kill parasites,which leads to the increasingly culturing environment and frequent occurrence of various diseases.The river crab "shuibiezi" disease has been a frequent outbreak since 2015,and the cause is unknown.The laboratory research found that the expression levels of glutathione transferase,carboxylesterase,and cytochrome P450 in diseased crabs increased.It is speculated that the,"shuibiezi" disease of crabs may be related to pesticide residues.To further study the relationship between pesticide residues and "shuibiezi" disease,the following work is mainly carried out in this paper:1.Molecular cloning and sequence analysis of Es-GSTs genes in E.sinensisThe full-length sequences of three Es-GSTS were obtained by rapid amplification of cDNA ends(RACE)from the Chinese mitten crab,named Es-GSTl、Es-GST2 and Es-GST3.Es-GST1 was 1166 bp in length,including a 48 bp 5’untranslated regions(UTR),473 bp 3’UTR,645 bp ORPF encoding 214 amino acids.Es-GST2 was1794 bp,including a 183 bp5’UTR,957bp3’UTR,654bp ORF,encoding 217 amino acids.Es-GST3 was 986 bp,including a 135 bp 5’UTR,176 bp 3’UTR,675 bp ORF,encoding 224 amino acids.Multiple alignments showed that all the three Es-GSTs contained a typical GST-N domain,a GST-C domain,and a G-binding site(FPNLPYIDGD).Among them,Es-GST2 and Es-GST3 exhibited unique Mu-loop domains,GEAPDYD and GDPPEYD.Amino acid sequence alignment revealed that the amino acid sequences of Es-GSTl、Es-GST2 and Es-GST3 had high homology with those of Procambarus clarkii,Penaeus monodon and Procambarus clarkii,was 56.36%、79.26%and 55.29%.The phylogenetic tree showed that Es-GSTl was clustered with Procambarus clarkia,further,Es-GST2 was clustered with Penaeus monodon and Penaeus vannamei.Finally,Es-GST3 was clustered into one branch with Penaeus japonicus and Penaeus vannamei.2.The expression and GST activity of Es-GSTs under three pesticide stressesThe expression patterns of three Es-GSTs in the heart,gill,stomach,hepatopancreas,muscle,intestine,testis,ovary,and accessory gonads of E.sinensis were detected by RTqPCR.The results showed that there was no significant difference in the expression levels of three Es-GSTs between male and female crabs in nine tissues,and they had high expression in the hepatopancreas and muscle.RT-qPCR revealed that the expression levels of the three Es-GSTs genes in the hepatopancreas and muscle of E.sinensis exposed to β-cypermethrin,trichlorfon,and avermectin significantly changed relative to those of the control group.The results of enzyme activity showed that under the three pesticide stresses,the GST activities in the control group varied by a small extent at six time-points,while the GST activities in the experimental group were higher compared to the control group,and the enzyme activity in hepatopancreas is higher than that in muscle.3.Visual observation and histopathology of crab tissues after exposure to low-concentration of insecticidesUnder the long-term stress of exposure to low insecticides concentrations of βcypermethrin and trichlorfon(Set the concentration to be 10 times lower than the safe concentration obtained in the LD50 experiment:β-cypermethrin 8.52 × 10-9g/L,trichlorfon 5×10-5 g/L),the crabs ate significantly less,moved slowly and became slow response,the color of hepatopancreas shifted from lighter color to gray-white to white gradually.Histopathology of the hepatopancreas,the nucleus ruptured,the contents flowed out,several vacuoles have occurred.Histopathology of the gills,larger gill cavity,and increased blood cells have occurred.Histopathology of the muscles,the nucleus was significantly marginalized,numerous vacuoles have appeared,the muscle fiber was raptured.Histopathology of the myocardium,the nucleus was exuded,many vacuoles have appeared and myocardial cell space was increased.These symptoms are very similar to those of the crabs suffering from "shuibiezi",suggesting that pesticide residues are closely related to the occurrence of "shuibiezi".4.Expression and enzyme activity analysis of Es-GSTs in E.sinensis under low concentrations of trichlorfon and β-cypermethrin under long-term stressRT-qPCR revealed that the expression levels of the three Es-GSTs genes in the hepatopancreas of E.sinensis exposed to β-cypermethrin and trichlorfon significantly changed relative to those of the control group.The results of enzyme activities showed that under the two pesticide stresses,the enzyme activity of the control group remained unchanged,while the enzyme activity of the experimental group was always inhibited,but there was almost no significant difference compared with the control group.