The Resistance Monitoring And Mechanisms Of Common Insecticides In Apolygus Lucorum (Meyer-Dür)

As the ever minor pest in cotton field, mirids became primary and also brought damage to various crops because of the large-scale plantation of Bt cotton. The control of the mirids has been dependent on the application of insecticides. The susceptibility of chlorpyrifos, malathion, methomyl, lambda-cyhalothrin, imidacloprid, and endosulfan to A. lucorum was monitored. The activities of the detoxification enzymes were measured in six field populations. A lambda-cyhalothrin-resistant strain of A. lucorum was selected and its biochemical and molecular basis of resistance was evaluated. Lastly, the resistant mechanisms towards lambda-cyhalothrin in Binzhou population were also investigated. These results will be helpful for the retional application of insecticides in A. lucorum.1. The resistance monitoring of common insecticides against A. lucorumBased on the suspectibility baseline, resistance to chlorpyrifos, malathion, lambda-cyhalothrin, endosulfan, methomyl, and imidacloprid was monitored by glass-vial bioassay at the diagnosis dose (LD99) in A. lucorum populations collected from six areas within Hebei, Shandong, Henan and Anhui province during 2013 and 2014. In 2014, Compared to the susceptible strain, the mortalities at the diagnosis dose (LD99) of lambda-cyhalothrin, imidacloprid, methomyl, and endosulfan within the SD-BZ population were 45%,60%,67% and 8%, respectively. This result suggested that the resistant individuals existed in SD-BZ population. For HB-QX, AH-WJ, AH-WW, and HN-XH population in 2014, the mortalities at the diagnosis dose (LD99) of endosulfan were about 50%, suggesting some individuals developed resistance towards endosulfan.The toxicities of common insecticides in six field populations were determined by a topical treatment technique. Compared to SS strain, six populations in 2014 had a higher resistance ratio to malathion and endosulfan than those in 2013. For chlorpyrifos in 2014, AH-WW population exhibited 7.8-fold resistant ratio compared to SS strain. SD-BZ population showed the resistance level of 29.9-fold towards lambda-cyhalothrin when compared to SS strain, while other field populations revealed the resistant ratios of 1.3 to 2-fold. For methomyl resistance, the ratios of six field populations between 2013 and 2014 were almost the same. Compared to SS strain, six populations in 2014 showed a higher resistance ratio to imidacloprid than those in 2013.The detoxification enzyme activities of field populations were measured by dynamics method. Compared to SS strain, the frequenties of field population individuals showing high CarE and GST (600-800 mOD/(min-pest)) were higher; the frequency of the acetylcholinesterase activity with more than 18 mOD/(minpest) was also higher in field populations than SS strain, suggesting the field populations with higher resistance had elevated enzyme activities.2. Characterization and alternative splicing of sodium channel gene of A. lucorumThe full-length sodium channel gene named AIVSSC was obtained by RT-PCR and rapid amplification of cDNA ends method (RACE). The full length of the cDNA of AlVSSC is total 6858bp, which contains a 6,087bp ORF (GenBank accession number: KR139855) encoding 2028 amino acid residues,523bp 5’UTR and 248bp 3’UTR respectively. A1VSSC protein possesses all predominant characteristics and conserved sequences of the sodium channel. Six different optional exons (A, B, C, D, E, F) and two mutually exclusive exons (K/L) were detected in AlVSSC transcripts.3. The analysis about mRNA level of gene associated with resistance against lambda-cyhalothrinThe relative expression levels of seven P450 monooxygenase detoxification enzyme genes were tested using quantitative real time (RT)-PCR. The results showed that four genes, CYP6X2, CYP6JB1, CYP6HM1, and CYP395H1, were found to be overexpressed in the resistance selection process by lambda-cyhalothrin (ranging from 2.5 to 21.6-fold). Among these genes, CYP6X2 was found to be overexpressed obviously with 21.6-fold. The mRNA level of CYP6X2 could be induced by the dose of LD20 lambda-cyhalothrin in SS strain and F11 generation of RR strain with 1.8 and 1.6-fold, respectively.4. The expression of CYP6X2, CYP6JB1, CYP6HM1, and CYP395H1 by Bac-to-Bac Expression SystemThe proteins of CYP6X2, CYP6JB1, CYP6HM1, and CYP395H1 associated with the resistance towards lambda-cyhalothrin in A. lucorum were expressed by Bac-to-Bac Expression System. The existence of expressed proteins was examined by Western Blot technology. The O-de-ethylation of 7-ethoxycoumarin (ECOD) activities of these four expressed proteins were 15.46±5.06,95.3±7.4, 61.94±10.48, and 148.8±8.54 pmoles 7-hydroxycoumarin/min/mg protein, respectively. These results could apply the basis for the metabolim of lambda-cyhalothrin.5. The analysis of the 5’flanking promoter region of CYP6X2The 5’flanking promoter region of CYP6X2 with the length of 1916 bp was obtained by genome walking kit. The potential bingding sites of transfactor were predicted by online software. The possible transfactor like HSF, Dfd, Bcd, GATA, GCM, and XRE-AhR were predicted to bind with the 5’ flanking promoter region of CYP6X2. Different length of promoter region of CYP6X2 was constructed into pGL-4.1 basic vector. The luciferase activity of the promoter region of CYP6X2 with the length of 1820 bp was highest compared to other length.6. The lambda-cyhalothrin resistant mechanism underlying SD-BZ populationThe activities of carboxylesterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase enzymes were measured between SS strain and SD-BZ population. Compared with SS strain, no significant difference (p>0.05) of the detoxify enzymes activities was found in this resistance population. One single point mutation L1015F in the AlVSSC was found only in the Shandong population. The frequencies of wild homozygote, mutant homozygote, and mutant heterozygote were 33.87,53.23 and 12.9%, respectively. Besides, the different genotypes at the L1015F locus of SD-BZ population were in Hardy-Weinberg equilibrium.7. The resistant mechnisms towards organophosphorus insecticides in Cangzhou, Hebei populationA topical bioassay method was performed to evaluate the toxicities of chlorpyrifos and malathion towards field-collected population of A. lucorum from Cangzhou, Hebei province. For chlorpyrifos and malathion, the resistance ratios was 10.7 and 15.5-fold respectively compared to a susceptible strain. A comparison of the detoxifying and target site acetylcholinesterase genes suggested that the mutation Ala216Ser within AlAChEl was most likely involved in organophosphorus resistance in A. lucorum. For HB-CZ and SD-BZ population, the frequency of mutant homozygote was 100 and 80% respectively. No mutant heterozygote genotype was found in both field population.