Biochemical And Molecular Toxicological Characterization Of GABA Related Enzymes From Bactrocera Dorsalis (Hendel)

The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera, Tephritidae) is one of the most important agricultural pests in many countries and regions. This pest has developed resistance quickly owing to its short life cycle, strong reproductive capacity, and unreasonable usage of the insecticides in the field. At present, a increasing number of researches about the monitoring, mechanism and management of resistance in the B.dorsalis were carried out focusing on the biochemical toxicology of detoxifying enzymes (such as P450s, GSTs, CarEs, etc.) and the molecular biological characteristics of target receptors (AChR). However, as an important target of avermectin and cyclodienes, such as dieldrin, little is known about the y-aminobutyric acid (GABA) receptor and characteristizationof its related enzymes in B. dorsalis.This research aimed to study the characterization of GABA receptor and the related enzymes through gene cloning, expression and analysis of enzyme activity. The susceptibility of Fuzhou population of the B.dorsalis to avermectin was determined through the local drop method. On this basis, the effect of avermectin on activitiy of y-aminobutyric acid transaminase and glutamic acid decarboxylase were detected, respectively. The genes encoding GABA receptor were cloned, and the expression mode in response to avermectin stimulation was analyzed. The main results obtained were as follows:1Determination of the susceptibility of the B. dorsalis to avermectinThe susceptibilities of Fuzhou (FZ) population of the B. dorsalis to avermectin were determined in the laboratory through the local drop method, and then the result was compared with other four geographical populations (Dongguan (DG), Guangzhou (GZ), Haikou (HK) and Kunming (KM) population). Bioassay results (24h) showed that the KM population was the most susceptible to avermectin, and DG population was found the least susceptible. The susceptibility KM and FZ populations were significantly different from other three populations. The sensitivity after72h was determined based on the function characteristics of avermectins, and LD50was only0.475ng/fly.2Biochemical and toxicological characterization of GABA related enzymes in B. dorsalis2.1GABA content determination The effect of avermectin treatment on GABA content in B. dorsal is was measured through HPLC. The content of GABA was increased after treated by avermectin (LD10,24h), but the change was not significant (1.13fold, P>0.05), while a significant increase was detected after treated by LD50(24h)(1.34fold, P<0.05). It was speculated that B.dorsalis coped with the stress of abamectin through maintaining high GABA levels.2.2Activity assay of GABA-T and GADThe activities of GABA-T and GAD, which associated with GABA content, was tested via micro-plate reader. The change of the GABA-T activity was slight, while the activity of GAD increased significantly, which was positively correlated with the dose and treatment time of avermectin. It was consistent with the changes of GABA content, tbat the increased GAD activity led to the increase of GABA content in B. dorsalis.3Molecular biological characteristics of GABA related enzymes in B.dorsalis3.1Molecular cloning and sequence analysis of B. dorsalis GABA receptor related genesTwo complete cDNA sequences and three partial sequences encoding GABA receptor were isolated using primers designed against the sequences identified from transcriptome data of B. dorsalis through RT-PCR and RACE technology. These sequences have been deposited in GenBank with the following accession numbers: BdGAP (JQ796071.1), BdGD1(KC763804), BdGAF (KC763801), BdGBR (KC763802) and BdSCD (KC763803). Proiparam and Scanprosite were used to predict the physicochemical properties of proteins, and MEGA5.0was used to construct the phylogenetic trees to determine the relationship of these genes with other insects’ GABA receptor downloaded from GenBank.3.2mRNA expression profiles of B. dorsalis GABA receptor related genesExpression profiles of four GABA receptor related genes in different developmental stages (egg, larva, pupa and adult), different tagmata (head, thorax and abdomen) and stimulated by abamectin were analyzed through qPCR. The highest expression of GAF was found in egg, while both GBR and GDI were in larva, and SCD was in adult The relative expression levels of GBR and SCD in head were significantly higher than that in breast and abdomen, and the expression of GD1in abdomen was the highest, while there was no expression difference of GAF in different tagmata. No significant change of expression of SCD was found after stimulated by abamectin, but the expressions of GAF, GBR, and GD1were significantly up-regulated. The expression levels of SCD, GAF and GBR were significantly up-regulated after24h exposure to different doses of abamectin (LD10and LD50, respectively) and increased with the dosages; the expression level of GD1increased after exposure to LD10of abamectin, but declined after exposure to LD50.The research analyzed the mechanism of the oriental fruit fly responsing to abamectin with methods in biochemistry and molecular biology areas. The results will improve the understanding of insecticide metabolism and resistance in this insect, suppling basis to retard and control the resistance development.