Molecular Detection Of Pesticide Resistance And Development Of Potential Control Strategies In The Colorado Potato Beetle, Leptinotarsa Decemlineata (Say)

The Colorado Potato beetle, Leptinotarsa decemlineata (Say)(Coleoptera, Chrysomelidae), is an international quarantine pest. The beetle invaded China in the1990s from Kazakhstan. Since then, it has spread eastward, and currently distributed through most of northern Xinjiang.Defoliation by L. decemlineata is a serious threat to potato and eggplant crops in northern Xinjiang. Insecticide treatments are currently indispensable for and effective on L. decemlineata control. Unfortunately, this pest has remarkable ability to develop insecticide resistance to virtually every chemical that has ever been used against it. Therefore, it is urgent and essential to estimate insecticide resistance in L. decemlineata in northern Xinjiang for proper choice of insecticides, and to develop potential management strategies.1. The molecular detection of the resistance to pesticides in L. decemlineataA modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVsscl genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were17.6%and14.7%,50.6%and42.2%,49.9%and41.7%,51.3%and41.4%, and44.8%and47.4%; to pyrethroids (L1014F mutation) were5.8%and8.7%,36.1%and27.0%,41.8%and24.8%,12.2%and9.7%, and7.9%and10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. No I392T point mutation in the AChE was detected by RT-PCR among18individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVsccl are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region.2. Efficacy of endosulfan and joint toxic action of its mixtures against L. decemlineata Insecticides with different modes of action may serve as likely replacements. Endosulfan is GABA-gated chloride channel blocking insecticides. Regarding stomach toxicity of the compound, adult beetles were less sensitive than2nd-,3rd-, and4th-instar larvae, suggesting that the appropriate timing for spraying is the early larval stage. Mixtures of endosulfan and phoxim at1:24ratio, and of endosulfan and isocarbophos at1:72and1:288ratios, significantly increased toxicity in a field population. The combination indices were significantly below1at both LD50and LD90levels, revealing synergistic effects. Our results demonstrated that endosulfan could be applied alone and may also be used in binary mixtures to control pyrethroid-resistant field populations. These findings may have considerable practical implications for L. decemlineata resistance management.3. Molecular cloning and race of genes encoding cytochrome P450monooxigenase from L. decemlineataBased on a transcriptome from L. decemlineata,38cytochrome P450fragments were cloned and named by Dr. Nelson. This will facilitate the future research on this topic. To comprehensively understand the cytochrome P450monooxigenase families, we integrated the data from NCBI and the results obtained previously in our group. In total, there were93cytochrome P450monooxigenases in L. decemlineata. All of them belonged to CYP2, CYP3, CYP4, or mitochondrial CYP450clades. CYP2clade had CYP18, CYP305, CYP306and CYP307families and only1member was found in each of the families. In CYP4clade, there were20,3,2and1members in CYP4, CYP350, CYP412and CYP413families, respectively. In CYP3clade,23,17,7and1genes were found in CYP6, CYP9, CYP345and CYP347families respectively. In mitochondrial CYP450clade,7,1,2,1,1,1,1and1were cloned in CYP12, CYP49, CYP301, CYP302, CYP314, CYP315, CYP334and CYP353families respectively. Three new proteins named CYP412al, CYP412a2and CYP413al were discovered. Full-length cDNAs of CYP18al CYP306a1, CYP307a1, CYP302a1, CYP314al and CYP315al were cloned using RACE technology. All of the6members were presumed to be involved in ecdysone metabolism.4. RNAi-based functional determination of the6genes encoding cytochrome P450putatively involving in ecdysone metabolismWe found that ARF1and RP18can be used as reference genes when qPT-PCR was performed to quantify relative expression level of any genes. By ingesting E. coli expressing dsRNA by the1st instar larvae, the genes encoding CYP18al, CYP306al, CYP307al, CYP302a1, CYP314al and CYP315al were successfully interferred. This seriously affected survival rate, body weight, pupation rate and development duration. Topical application of20-hydroxyecdysone or halofenozide (an ecdysone agonist) could partially rescue the negative effects by RNA interference of CYP306a1, CYP307a1, CYP302a1, CYP314a1and CYP315a1, genes encoding five enzymes for20-hydroxyecdysone biosynthesis, but enhance the negative effect of RNA interference of CYP18a1, which encoded an enzyme for20-hydroxyecdysone degratation. Our results demonstrated that ingesting E. coli expressing dsRNA by the1st instar larvae could successfully interferred target genes, and indicated that CYP18a1, CYP306a1, CYP307a1, CYP302a1, CYP314a1and CYP315a1should be responsible for ecdysone metabolism in L. decemlineata.