Biochemical And Molecular Toxicological Characterization Of GSTs From Oriental Fruit Fly,Bactrocera Dorsalis (Hendel)(Diptera:Tephritidae)

The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera:Tephritidae), is a severe pest of fruit and vegetable pests. Based on its significantly economic importance, researches on population genetic structure, pheromone products, and chemical control were conducted in recent years, while B. dorsalis has generated heavy resistance to insecticides due to long-term and unreasonable using chemical control. Efforts to control this pest often result in development of insecticide resistance. It has been shown to develop resistance and cross-resistance to various different classes of insecticides, including organophosphates, pyrethroids, and carbamates. Until now, the mechanism of insecticides resistance of B. dorsalis has focused on target enzyme:the biochemical and molecular characterization of acetylcholinesterase. However, very little is known about the characteristics of detoxification enzymes of oriental fruit fly, despite their significance in the metabolic of xenobiotics and insecticide resistance. Glutathione S-transferases (GSTs, EC2.5.1.18) are a diverse super family of enzymes with a range of catalytic functions, including cellular protection from reactive oxygen species, reductive maintenance of thiolated proteins, prostaglandin synthesis, and glutathione conjugation of endogenous and exogenous ligands. The GSTs from Delta and Epsilon family are insect specific, which have important roles in phase Ⅱ detoxification of several chemical insecticides classes, including organophosphates, chlorinated hydrocarbons, and pyrethroids.The current study was aimed at the insect adaption mechanisms environment stress. We comprehensicely studied the purification, biochemical and toxicological characterization of GSTs, molecular cloning and sequences analysis of GSTs genes, GSTs genes expression profiling, and prokaryotic expression of GSTs genes. The results would help to clarify the molecular toxicology mechanism of to molecular toxicology of B. dorsalis to insecticide resistance, which could also act as a molecular diagnostic technique to monitor the resistant level of field populations. Our results will provide a theoretical basis for the sustainable management of B. dorsalis, and enrich and develop the scientific theoretical study system of insect pests resistance to insecticides. The main results are as follows:1. Purification, biochemical and toxicological characterization of GSTs from B. dorsalis1.1Purification, biochemical and toxicological characterization of GSTs from four field populations of B. dorsalisIn this study, GSTs were purified from four field populations of B. dorsalis with different insecticide susceptibilities by glutathione-agarose affinity chromatography. The populations were collected from Dongguan (DG) and Guangzhou (GZ) of Guangdong Province, Haikou of Hainan province (HN), and Kunming of Yunnan province (YN), China. Differences in GST characteristics among the four populations were studied using purified enzyme samples through comparative SDS-PAGE, kinetic, and inhibition experiments. The specific activities of the purified enzymes were similar, but the purification yield of the GZ population (31.54%) was the lowest. SDS-PAGE analysis showed only one band at approximately23kDa for these four populations. Kinetic analyses showed that the affinities of the purified GSTs from the GZ and YN populations for1-chloro-2.4-dinitrobenzene (CDNB) were much higher than those of GSTs from the other two populations, while the HN population had the highest catalytic capability in terms of Vmax value. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all four populations. Inhibition kinetics showed that ethacrynic acid, diethyl maleate, tetraethylthiuram disulfide, curcumin, bromosulfalein, and beta-cypermethrin had excellent inhibitory effects on GSTs in the four populations of B. dorsalis, but the low inhibitory effects of malathion and avermectin did not differ between populations. These results suggest that GSTs may have a role in detoxification of (3-cypermethrin in B. dorsalis.1.2Purification, biochemical and toxicological characterization of GSTs from three development stages of B. dorsalisIn this study, GSTs were purified and characterized from three development stages (third larva, pupa, adult) of B. dorsalis by glutathione sepharose4B affinity chromatography. The chromatography elutions are showed the highest specific activity of GSTs between third to fifth fraction. The both total activity and specific activity of adult showed higher values than those of third larva and pupa, with370.76nmol.min-1and16.91nmol.min-1.μg-1, respectively. SDS-PAGE analysis showed only one band at approximately23kDa for these three development stages. In both CDNB or GSH as substrate, the GSTs Km value of larval stage were significantly lower than those in the pupa and adult stages, while there were no significant difference among three development stages of GSTs Vmax. The optimum temperature for CDNB conjugation was37℃, and the optimum pH was7.5in all three development stages. The I50values of ethacrynic acid (1.15μM) and diethyl maleate (0.60μM) against the adult stage were significantly lower than those of the other stages. The I50value of bromosulfalein did not differ significantly among the three development stages. The I50value of disulfiram among the pupa and adult stages were significantly higher than that of third larva, while there were no significant difference between pupa and adult stages. The I50value of curcumin (101.78μM) against the adult stage was significantly higher than those of the other stages. The I50values of β-cypermethrin against the pupa (0.90mM) and adult (0.78mM) stages were significantly higher than that of larva stages. The low inhibitory effects of malathion and avermectin did not differ between development stages. The results suggested that B. dorsalis was more sensitive to the insecticide β-cypermethrin in the larva stage than other two stages.2. Molecular cloning and sequences analysis of GST genes from B. dorsalisBased on the transcriptome data,18novel GST genes were cloned from B. dorsalis using the RT-PCR and RACE techniques. Molecular characterizations of the putative proteins have been predicted by Protparam software. According the location with the cell, these18GSTs genes were classified into17cytosolic and1microsomal. Phylogenetic analysis of these17cytosolic GSTs deduced from their cDNAs revealed16GSTs that belong to five different cytosolic classes, including4in delta,8in epsilon,2in omega,1in theta, and1in zeta, based on their sequence similarities to other insect GSTs, particularly those from Drosophila melanogaster, Anopheles gambiae, and Aedes aegypti. The remaining one GST was unclassified due to the lack of homologies to the currently known class. The nomenclature of these B. dorsalis GSTs cDNA and their deduced amino acid sequences have been deposited in Genbank with the following accession numbers:BdGSTdl (JQ690090), BdGSTd2(JN003593), BdGSTd5(JN792452), BdGSTd6(JN792453), BdGSTel (JN003588), BdGSTe2(JN003589), BdGSTe3(JQ690091), BdGSTe4(JN003590), BdGSTe5(N003591), BdGSTe6(JQ690092), BdGSTe7(JN003592), BdGSTe9(JQ690093), BdGSTol (JQ690094), BdGSTo2(JQ690095), BdGSTtl (JQ690096), BdGSTz2(JQ690097), and BdGSTul (JN003587). The percentages of deduced amino acid identities were47.6-57.9%among the four delta GSTs,31.8-54.9%among the eight epsilon GSTs,85.4%between the two omega GSTs, but only12.4to30%among different classes of the GSTs in the oriental fruit fly GSTs. Furthermore, several conserved amino acid residues which represent the catalytic pocket and binding site were analyzed by sequence alignment. Meanwhile, the full-length cDNA of microsomal GST gene BdGSTml was identified and cloned from B. dorsalis using RACE technique. The Genbank accession number was JQ690098. In addition, BdGSTm1was closely related to DmGSTm-NP524696with91%amino acid sequence identity. The deduced amino acid sequence included one insect typical motif of the microsomal GSTs family, was D-P-X-V-E-R-V-R-R-A-H-X-N-D-X-E-N-I-L-P, based on their sequence similarities to other diptera insect microsomal GSTs. At the same time, insect microsomal GSTs amino acid sequences were shorter, usually only about150amino acids.3. GST genes expression profiles from B. dorsalis3.1Developmental stages expression profiles of GST genesThe developmental stages expression profiles of18GST genes from B. dorsalis were analyzed using a-Tubulin as an internal control gene. The developmental stages included egg, three different larval instars (first-, second-, and third), pupa, and adult. The results showed that the relative expression level of BdGSTel was the only overexpressed gene in the egg developmental stage, which suggested that it might be associated with regulation of juvenile hormone and ecdysone in egg. Higher expression levels of six GST genes (BdGSTd2, BdGSTd5, BdGSTe5, BdGSTe9, BdGSTtl, and BdGSTz2) were observed at larval stages compared with adulthood, implies it may play an important metabolic role in the larval stages. The expression level of four Epsilon GST genes (BdGSTe2, BdGSTe3, BdGSTe4, and BdGSTe6) were increased during the development of fruit fly, which indicates that the detoxification capacity also increase in the ontogeny of B. dorsalis.3.2Expression profiles of GST genes among different tissuesThe relative quantitative of mRNA expression levels of18GST genes in three tissues including midgut, fat body and Malpighian tubules were analyzed using a-Tubulin as reference gene. In the midgut tissue, the expression of three insect-specific family GST genes (BdGSTd5, BdGSTe3, and BdGSTe9) were the highest, suggesting that these genes may play an important role in the detoxification of exogenous compounds. Other GST genes were not expressed in the midgut, may play a similar functional roles in other tissues. Four GST genes(BdGSTd6, BdGSTe4, BdGSTe6, and BdGSTz2) in the fat body tissue were relatively the highest expression level, indicating that these genes may be involved in the detoxification of exogenous compounds or protect the body from oxidative stress. The relative mRNA expression of BdGSTdl, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe7, and BdGSTu1were higher in the Malpighian tubules tissue than the other tissues. The specific role of GSTs in Malpighian tubules is still not clear, but it could be involved in the enhanced excretion of Malpighian tubules.3.3Expression profiles of GST genes induced by three insecticidesTo determine the induction responses of GST genes, three insecticides malathion, avermectin, and β-cypermethrin were chosen in this study. Based on the bioassay, the flies were induced by the insecticides in two different regimes:time treatment (12,24,36, and48h) and dose treatment (LD10, LD50, and LD80). qPCR with a-Tubulin as the reference gene was used to determine the relative expression quantity of GST genes. Five GST genes (BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTm1) transcripts showed the highest after12h malathion induction; BdGSTd2and BdGSTo1were reached the highest after24h; and other eight GST genes(BdGSTd1, BdGSTel, BdGSTe2, BdGSTe3, BdGSTo2, BdGSTtl, BdGSTz2, and BdGSTu1) reached the peak48h after malathion induction. The relative expression quantities of ten GST genes (BdGSTd1, BdGSTd5, BdGSTd6, BdGSTe2, BdGSTe4, BdGSTe5, BdGSTe6, BdGSTo2, BdGSTtl, and BdGSTu1) were increased significantly after exposure to sub-lethal dose (LD10) malathion for24h. These GST genes may participate in the metabolic processes to malathion of B. dorsalis.In the avermectin induction, the expression of BdGSTe6and BdGSTml were significantly enhanced after12h, meanwhile these six GST genes(BdGSTd1, BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, and BdGSTe9) transcripts were reached the peak24h later, seven GST genes(BdGSTd2, BdGSTd5, BdGSTd6, BdGSTe4, BdGSTe5, BdGSTe6, and BdGSTml) were the highest after induce to lethal dose (LD50) avermectin for24h. The results suggest that these GST genes may have a role in detoxification of avermectin in B. dorsalis.Similar to the above insecticides, after the12h β-cypermethrin induction, the expression of three GST genes(BdGSTe2, BdGSTe5, and BdGSTe6) were significant higher; meanwhile these six GST genes (BdGSTe4, BdGSTe9, BdGSTo2, BdGSTtl, BdGSTu1, and BdGSTm1) expression were reached the peak36h later, four GST genes (BdGSTd1, BdGSTe4, BdGSTe7, and BdGSTtl) were up regulation after induce to lethal dose (LD10) P-cypermethrin for24h, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.3.4Expression profiles of GST genes among different strainsIn order to verify whether GSTs are over-expressed in resistant fruit flies, qPCR with a-Tubulin as the reference gene was employed to determine the relative expression quantity of18GST genes in three different strains of B. dorsalis. As indicated by qPCR, three GST genes (BdGSTd5, BdGSTe9, and BdGSTz2) transcripts in malathion resistant strain were significantly higher than susceptible strain, while the expression of BdGSTel, BdGSTe2, and BdGSTtl were significantly lower than susceptible strain, which implied that these GST genes were possibly involved in B. dorsalis malathion resistance. The relative expression levels of these eight GST genes (BdGSTd6, BdGSTel, BdGSTe2, BdGSTe5, BdGSTe6, BdGSTe7, BdGSTtl, and BdGSTm1) were significantly higher in β-cypermethrin resistant strain than susceptible strain, suggesting that these GST gene may have a role in detoxification of β-cypermethrin in B. dorsalis.4. Heterologous expression of BdGSTe5in Escherichia coliUsing the double digestion of Ndel and HindⅢ restriction enzymes and the DNA recombination technology, the expression vector for BdGSTe5was constructed based on pET28a (+) vector. After IPTG induction to E. coli, SDS-PAGE analysis showed only one specific band at approximately27kDa for the recombinant plasmid BdGSTe5-pET28a (+). According to the protein molecular weight, the expression of fusion protein molecular weight was about26kDa. Because of6x His-tag and flanking fragment accounting for1kDa, therefore, the specific band was consistent with the expected size of fusion protein. BdGSTe5-pET28a (+) recombinant protein was purified and confirmed by Ni and DEAE column.In summary, the purification, biochemical and toxicological characterization of GSTs of B. dorsalis were studied by glutathione-agarose affinity chromatography; based on the transcriptome data,18novel GST genes were cloned from B. dorsalis using RACE technique; GST genes expression profiles were studied using qPCR technique from B. dorsalis including developmental stages, tissues, insecticides inductions, and different strains, furthermore, the expression vector for BdGSTe5was constructed and expressed. These results will not only provide great insights into exploringthe functions of the oriental fruit fly GSTs system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms of oriental fruit fly to environment. Meanwhile, the results would also enrich and develope the scientific theoretical study system for the resistance mechanism of other insect pests.