| Carboxylesterases (CarEs) as one of the predominant metabolic enzyme familys in insect play the primary role in degredation and sequestration of a broad suit of endogenous and exogenous ester containing compounds. CarEs are frequently implicated in the resistance of insects to different insecticides. Locusta migratorria manilensis (Meyen) is an important agriculture pest in the world. In the past years, L. migratorria manilensis (Meyen) had been controlled by applying massive insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of resistant strains. In 2004, Ma et al. firstly reported OP resistance of L. migratoria manilensis in the world. Yang et al. demonstrated that the OP resistance of L. migratorria manilensis was mediated by the increased activities of CarEs.In our present study, the aim of research was to elucidate the molecular mechanism of esterase-mediated malathion resistance in locust.25 expressing sequence tags (ESTs) of CarEs were obtained by bioinformatics methods from the database of ESTs in L. migratoria manilensis according to the conservative motifs of CarEs in insects.12 CarE gene fragments potentially correlated with OP resistance were screened out by comparing the CarEs expression level between a laboratory-susceptible strain (LS) and a field-resistant population (FR) via real-time quantitative PCR (statistically significant differences, P<0.05 by t-test). The tissue and head (including foregut, midgut, hindgut, gastric caecum, fatbody, Malpighian tubules, integument and head) expression patterns of 12 CarE gene fragments between the FR and LS were performed by regular RT-PCR, the results indicated that 6 CarE genes (LmCarE6, LmCarE9, LmCarEll, LmCarE19, LmCarE24, LmCarE25) mainly expressed in gastric caecum, the expression levels of 6 CarE genes in FR are higher than those in LS. Only a gene fragment (LmCarE20) predominantly expressed in head. The remaining CarE genes differently expressed in several selected tissues.Developmental expression of 12 CarE gene fragments in LS showed that all of 12 CarE gene fragments consistently expressed through six stages including 1st to 5 th instar nymphs and adults in either gastric caecum or whole body.The full length sequence of 2 CarEs were gained. Insect carboxyl/cholinesterases have been partitioned 14 clades by the phylogeny, denoted A to N. Carboxyl/cholinesterases associated with insecticide metabolic detoxification clustered into A, B and C clades. In this research, the other known 31 insect sequences from three clades (A, H, and N) were used to construct phylogenetic tree with LmCarE9 and LmCarE25 by neighbor-joining methods. Phylogenetic tree also demonstrates that the LmCarE9 and LmCarE25 are members of carboxylesterases and can be classified into subgroup A. Thus the two cloned locust CarEs are named as LmCarEAl (for LmCarE9) and LmCarEA2 (LmCarE25), respectively.Two representative genes (LmCarE9 and LmCarE25) were chosen for RNAi followed by RT-PCR and malathion bioassay. RT-PCR showed that the expression of both CarEs genes decrease at 12 h after injected dsRNA, the significant silence of both CarEs genes expression were detected at 24 h after injected dsRNA. However, the level of both CarEs genes expression reached original level at 72 h after dsRNA injection. Compared with ddH2O injection control, the susceptibility of locust to malathion was significantly decreased after injected dsRNA of LmCarE9 and LmCarE25. These results indicated significant roles of these CarE genes in conferring malathion resistance in the locust.In a word, these results will provide new insight to explore resistant molecular mechanism in locust, which will be benefit for locust resistant monitor and management in the field.
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