Molecular Characterization And Tissue Localization Of Carboxylesterase Genes In Locusta Migratoria

Locusta migratoria is one of the most damaging agricultural pests in our country. Insecticide resistance in Locusta migratoria due to extensive uses of chemical insecticides for its control has been reported. Insecticide resistance not only makes it difficult to control the locust, but also causes serious environmental problems. Carboxylesterase superfamily belongs to one of the three major metabolic detoxification enzyme systems in insects, which are often responsible for or contribute to insecticides resistence in L. migratoria. To further elucidate the roles of carboxylesterase genes in insecticide detoxification in L. migratoria, we searched the Locust EST library and transcriptome database and identified several cDNAs putatively encoding carboxylesterases. We subsequently sequenced and analyzed two full-length cDNAs and launched a series of molecular studies to characterize their properties and functions.1. Verification and analysis of cDNA sequence of carboxylesterase genes in L. migratoriaWe sequenced and verified full-length cDNA sequences of LmCarE26and LmCarE27using PCR. The LmCarE26cDNA contains2176nucleotides with an open reading frame (ORF) of1614nucleotides that encodes538amino acid residues. The LmCarE27cDNA consists of1520nucleotides with an ORF of1410nucleotides that encodes470amino acid residues. However, no polyadenylation signal was found in either of the two carboxylesterase cDNAs. The deduced amino acids of both carboxylesterase genes contained conserved catalytic sites. The predicted molecular masses of their deduced proteins, LmCarE26and LmCarE27, were59.8and52.2kDa, respectively. The estimated isoelectric points (pIs) of LmCarE26and LmCarE27were about6.22and5.98, respectively.2. Tissue-and developmental stage-dependent expression patterns of two carboxylesterase genes in L. migratoria Tissue-and developmental stage-dependent expression patterns of LmCarE26and LmCarE27were analyzed using real-time quantitative PCR. Our results showed that both carboxylesterase genes were expressed in nine different tissues including brain (BR), foregut (FG), gastric caeca (GC), midgut (MG), Malpighian tubeles (MT), hindgut (HG), fatbodies (FB), muscles (MU) and hemolymph (HE). Although both LmCarE26and LmCarE27were highly expressed in GC and MG, the highest expression for LmCarE27was in GC, whereas the highest expression for LmCarE26was in FB. Overall, the expression levels of these two genes were significantly higher in MG, GC, MT and FB than in HE, BR, FG, HG, and MU. With respect to the developmental stage-dependent expression patterns of LmCarE26and LmCarE27, we found that both genes were highly expressed throughout the developmentl stages. Although LmCarE26showed an expression pattern similar to that of the juvenile hormone (JH) titer, we can not claim that LmCarE26is a JH esterase gene based on our current studies.3. Different roles of carboxylesterase genes in insecticide detoxification of L. migratoria.We peformed RNAi for each of the two carboxylesterase genes followed by bioassay with each of four insecticides including malathion, deltamethrin, chlorpyrifos and carbaryl. Mortality in malathion-, carbaryl-, and deltamethrin-treated locusts increased by31,15and29.5%after the injection of LmCarE26dsRNA as compared with those controls injected with GFP dsRNA followed by same insecticide bioassays. However, the injection of LmCarE26dsRNA did not increase with the susceptibility of the locusts to chlorpyrifos. In contrast, the injection of LmCarE27dsRNA did not significantly increase the susceptibility of the locusts to any of the four insecticides. All these results suggested that LmCarE26played an important role in the detoxification of malathion, carbaryl and deltamethrin, whereas LmCarE27might have other biological functions.4. Localization of carboxylesterase transcripts in L. migratoria We located LmCarE26and LmCarE27transcripts by using in situ hybridization. Both LmCarE26and LmCarE27were highly expressed apical and basal of the columnar cells in GC. These are important regions for food digestion and nutrition absorption, and for chemical metabolism by various enzymes such as carboxylesterases. These results are consistent with our PCR analysis showing that GC was one of a few main tissues with highest expression of LmCarE26and LmCarE27.In summary, we characterized molecular properties, evaluated the roles in detoxification of four commonly used insecticides and revealed tissue-specific expressions of LmCarE26and LmCarE27in L. migratoria. Our results are expected to help us develop better strategies for resistance management and effective control of this important insect pest.