Four Carboxylesterases From Chinese Mitten Crab Eriocheir Sinensis:cDNA Cloning,Tissue Expression,and Functional Analysis After Pesticide Treatment

Chinese mitten crab Eriocheir sinensis,also known as the river crab,is one of the most economically important crustacean cultivated in China.With the rapid increase of the E.sinensis aquaculture industry,its breeding scale and density are also expanding.Both abiotic and biotic stressors are intensifying in aquaculture.Numerous diseases have recently evolved and many pest control products have been used.In recent years,due to the increasing market demand of river crab,its breeding scale and density are also expanding.In addition,pesticides and algaecides are also widely used,which leads to the increasingly harsh breeding environment and frequent occurrence of various diseases.At the same time,in agricultural production,insecticides are used to kill crop pests.Pesticide residues may enter aquatic ecosystems via surface runoff,rainwater scour,and domestic wastewater.These inputs may also pollute aquaculture water sources.Pesticide residues might therefore cause various crustacean diseases.As a multi-functional family enzyme system,carboxylesterase can participate in signal transmembrane transduction,metabolic detoxification of organophosphorus insecticides and other pest control products,and lipid synthesis and decomposition.However,there are litter known about pesticide resistance mechanisms of Carboxylesterases in E.sinensis.In this study,full-length sequences of four ES-CXE genes from Chinese mitten crab were cloned and tissue-specific expression levels of ES-CXEs were analyzed.The expression level of four ES-CXEs genes and carboxylesterase activity change in hepatopancreas of E.sinensis after treatment with β-cypermethrin,trichlorfon and avermectin also were tested.The main results are as follows:1)Cloning of full-length ES-CXE cDNAsThe full-length cDNAs of four ES-CXEs were obtained by rapid amplification of cDNA ends(RACE)from the Chinese mitten crab,named ES-CXE1,ES-CXE2,ES-CXE3 and ES-CXE4,respectively.The full-length cDNA ES-CXE1 sequence obtained from the hepatopancreas of Chinese mitten crab by RACE was 2,446bp.It consisted of a 5’-untranslated region(UTR)of 150bp,a 3’-UTR of 526bp with a Poly-A tail,and an ORF of I,770bp.This ORF encoded589 amino acids.The full-length cDNA sequence of ES-CXE2 was 2,384bp.It consisted of a 5’-UTR of 72bp,a 3’-UTR of 536bp with a Poly-A tail,and an ORF of 1,776bp encoding 591 amino acids.The full-length cDNA sequence of ES-CXE3 was 1,889bp.It consisted of a 5’-UTR of 132bp,a 3’-UTR of 122bp with a Poly-A tail,and an ORF of 1,635bp encoding 544 amino acids.The full-length cDNA sequence of ES-CXE4 was 3,089bp.It consisted of a 5’-UTR of 282bp,a 3’-UTR of 799bp with a Poly-A tail,and an ORF of 2,028bp encoding 675 amino acids.Multiple alignments revealed that both ES-CXEs contained domains typical of the Carboxylesterases family proteins,including three amino acid residues of the catalytic triad serine(S),glutamic acid(E),and histidine(H)and a catalytic N-terminus region.Amino acid sequence alignment revealed that the amino acid sequences of ES-CXE1,ES-CXE2 and ES-CXE3 had high homology with those of Portunus trituberculatus,Penaeus vannamei,Pandalus shrimp,ranging from 40%to 50%,while the amino acid sequences of ES-CXE4 had high homology with Daphnia magna(39.42%).Phylogenetic tree analysis showed that the amino acid sequences of ES-CXE1,ES-CXE2 and ES-CXE3)were highly similar to those of Portunus trituberculatus,Penaeus vannamei,Pandalus shrimp,and preferentially clustered into one group.However,the ES-CXE4 amino acid sequence was similar to that of Daphnia daphnia,and it was clustered preferentially.2)Tissue distribution of ES-CXEsThe expression patterns of four ES-CXEs in the heart,gill,hepatopancreas,muscle,intestine,testis,ovary and accessory gonads of Eriocheir sinensis were detected by RT-qPCR.The results showed that there was no significant difference in the expression levels of four carboxylesterase genes ES-CXEs between male and female crabs in eight tissues,and they had expression in the hepatopancreas,muscle,testes,and accessory gonadal glands.However,its expression levels were low in the heart,gills,and ovaries.3)ES-CXEs expression pattern analysis after pesticide treatmentFluorescence quantitative PCR showed that the expression levels of the four ES-CXEs genes in the hepatopancreas of animals exposed to low doses of β-cypermethrin,trichlorfon and avermectin significantly changed relative to those of the control group,Therefore,treatment with these insecticides induced the expression of both ES-CXEs.4)Analysis of CXEs activity patterns after pesticide treatmentThe activities of CXEs at different time points after pesticide treatment were determined by spectrophotometry using the Carboxylesterase Activity Assay Kit.The enzyme activities of CXEs in the hepatopancreas determined following exposure to β-cypermethrin,trichlorfon and avermectin significantly were significantly higher than that of the control group.Therefore,treatment with these insecticides induced changes in carboxylesterase family genes activities.We believe that this study will provide insight on the pesticide resistance mechanisms associated with the CXEs in Chinese mitten crab.