Transcriptome Analysis And Insecticide Detoxification Of Carboxylesterase Genes In Locusta Migratoria

Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals. The genome sequence analyses has revealed a number of CarE genes in at least 10 insect species, including Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, Culex pipiens quinquefasciatus, Bombyx mori, Apis mellifera, Nasonia vitripennis, Tribolium castaneu, Acyrthosiphon pisum, Trialeurodes vaporariorum. These enzymes play important roles in detoxification of xenobiotics (including insecticides and secondary metabolism products of plants), degradation of pheromones and regulation of neurodevelopment. In our lab, bioassay and biochemical research indicated that field populations of L. migratorria had resistance to malathion and the OP resistance of locust was mediated by the increased activities of CarEs and upregulated mRNA expressions. Therefore, it is critical to conduct a comprehensive search of locust CarE genes and analyses of molecular characterization and insecticide detoxification of these genes. The main contents are as follows:1. Transcriptome analysis of cDNA sequences of L.migratoria CarE genesA total of 84 putative CarE cDNA fragments were obtained from L. migratoria transcriptome database by bioinformatics methods. We characterized a total of 39 full-length cDNAs with complete ORF putatively encoding different CarEs from L. migratoria. Our phylogenetic analysis indicated 71 locust CarEs were distributed in five clades, including clade A with orthopteran a-esterases (45 CarEs, including 20 CarEs with complete ORFs), clade D with integumental esterases (6 CarEs,3), clade E with β-esterases (17 CarEs,13), clade F with nonlepidopteran JHEs (1 CarE with complete ORF) and clade I with uncharacterized esterases (2 CarEs with complete ORFs). Physicochemical properties and conserved motifs analyses of deduced amino acid sequences of locust CarE genes were conducted by using ExPASy online software. The calculated molecular mass for the 39 CarEs ranged from 52.2 to 92.6 kDa. Most of CarEs are acidic or slightly acidic with isoelectric points ranging from 4.24 to 6.95. The signal peptide cleavage sites and potential N-glycosylation sites were predicted in 28 locust CarEs, but absent in the remaining 11 CarEs. Our research suggested most of locust CarEs have conserved motifs,34 CarEs have conserved catalytic triads (Ser-Glu/Asp-His; S-E/D-H).2. Analyses of tissue-and stage-dependent expression of locust CarE genesBased on phylogenetic analyses, eight CarE genes (LmCesAl, LmCesA2, LmCesAS, LmCesA20, LmCesDl, LmCesEl, LmCesFl and LmCesIl), representing five different clades (A, D, E, F and I), were selected for further analyses. The stage-dependent expression patterns of the eight CarE genes in L. migratoria were analyzed by RT-qPCR. The transcripts of all the eight genes were detectable in all developmental stages, indicating that the expression of these genes is ubiquitous in the locust. Except for LmCesIl, the high transcripts of the remaining genes were detectable in nymph stages and adults. However, these genes showed relatively low expressions in eggs as compared with other developmental stages. Tissue-dependent expression and in situ hybridization of two CarE genes showed that LmCesAl and LmCesA2 were mainly expressed in gastric caeca. The results of tissue-dependent expression indicated that LmCesA20, LmCesDl and LmCesI1 were predominately expressed in the muscles and hemolymph. LmCesA3 and LmCesEl were mainly expressed in the fat bodies and Malpighian tubules, whereas the highest expressions of LmCesFl were found in the foregut, hindgut and fat bodies, and its lowest expressions were in the gastric caeca and midgut.3、Effect of deltamethrin on CarE gene expression in L. migratoriaDeltamethrin is a kind of pyrethroid insecticides with merits of high insecticide activity, broad insecticide spectrum, rapid efficacy and crop safety. Thus, it will provide a theoretical basis for resistance risk assessment in L. migratoria to analyze mRNA expression of CarE genes in response to deltamethrin exposure. Locusts were exposed to different doses of deltamethrin and treated with deltamethrin for different periods of time in order to analyze the mRNA expression of CarE genes in response to deltamethrin. Results indicated deltamethrin induced mRNA expression of 5 CarE genes, including LmCesAl, LmCesA3, LmCesDl, LmCesEl and LmCesI1. These results suggest these genes may play an important role in the detoxification of deltamethrin and the development of resistance to this pesticide in L. migratoria.4、Effect of RNAi of eight CarE genes on susceptibility to insecticidesRT-qPCR analyses at different times (12,24 and 48 h) after the injection of dsRNA for each target gene showed significantly decreased transcript levels of LmCesA1 at 24 h (about 87%) and 48 h (about 86%) and of LmCesA2 at 24 h (about 97%) as compared with those in the controls. Our RT-qPCR analyses showed significantly decreased transcript levels of each CarE gene at 24 h after the dsRNA injections. The silencing efficiency was≥ 70% in the nymphs as compared with that in the controls at 24 h, indicating an effective silence of these target genes by RNAi. To investigate possible involvement of LmCarEs in detoxification of four commonly used insecticides (chlorpyrifos, malathion, carbaryl, and deltamethrin), the susceptibility of the dsRNA-injected locusts to each of these insecticides was assessed at 24 h after dsRNA injection. Results from our insecticide bioassays showed that the susceptibility of the nymphs to malathion increased when LmCesA20 and LmCesEl were silenced. The increased susceptibility of the nymphs to chlorpyrifos was detected when LmCesAl and LmCesA2 was silenced, respectively. These results suggested that partial a-esterases and β-esterases might play a significant role in detoxification of OPs in locust. The nymph mortalities in response to carbaryl treatment increased after the expression of LmCesFl and LmCesI1 were suppressed, indicating these two genes most likely to be involved in detoxification of carbaryl in L. migratoria.5、Constrcution of transgenetic Drosophila and study on its susceptibility to insecticidesHomozygous line of L. migratoria LmCesA2 transgenic Drosophila (P20-LmCesA2) was successfully constructed by using classical Gal4/UAS system. Compared with the parental lines of attP40 and hybrid offspring between attP40 and GAL4, bioassays showed that hybrid offspring with LmCesA2 expression showed higher malathion tolerance, the calculated ratios of LD50were 1.14 and 1.23, respectively. These results were consistant with those of RNAi, and indicated that LmCesA2 be most likely involved in detoxification of malathion in L. migratoria. The mRNA upregulation of LmCesA2 might improve malathion resistance risk in L. migratoria.6. Eukaryotic expression of CarE gene from L. migratoriaThe full-length cDNA of LmCesA2 was sub-cloned into FastBacHTA vector, and the recombinant vector pFast BacTM-LmCesA2 was transformed into DH10 competent cells to obtain the bacmid DNA Bacmid-LmCesA2. sf9 cells were transfected with the bacmid DNA Bacmid-LmCesA2 by using lipofectin 2000 to get recombinant baculovirous. The results of western-blot showed that a specific band with 60 kDa was detected in the cytoplasm of sf9 cells, which is consistent with the expected size of the fusion protein, and suggested LmCesA2 was successfully expressed in sf9 cells.Conclusion, we identified 84 cDNA sequences of locust CarE genes from the L. migratoria transcriptome database, including 39 CarE cDNAs with complete ORFs. The deduced amino acid sequences of 71 CarE cDNAs were clustered into five different clades in phylogenetic tree. Significant expansions of the CarE genes were found in clade A (45 orthopteran a-esterase genes) and in clade E (17 β-esterase genes). Eight CarE genes were selected for further analyses, such as tissue-and stage-dependent expression of CarE genes and analysis of deltamethrin induction. RNAi and bioassays showed different locust CarE genes may play key roles in detoxification of malathion (LmCesA20 and LmCesEl), chlorpyrifos (LmCesAl and LmCesA2) and carbaryl (LmCesFl and LmCesI1). Furthermore, we successfully constructed transgenetic Drosophila with CarE gene of L. migratoria and analyzed its insecticide tolerance and established eukaryotic expression system of locust CarE gene. These results are expected to reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust.