Research On Resistance Mechanisms To Indoxacarb And Metaflumizone In Plutella Xylostella

The diamondback moth, Plutella xylostella (L.) (Lepidoptera:Plutellidae), is one of the most serious pests on the cruciferous vegetables. Due to the high value of these brassicas and the short generation time (14-28 days) of P. xylostella, a wide range of pesticides has been frequently used, which lead to widespread cross-and multiple-resistance to many insecticides. According to APRD, the diamondback moth had developed resistance to at least 92 kinds of active ingredients.The voltage-gated para-sodium channel is the primary target for the neurotoxins DDT, pyrethroids and other insecticides, including indoxacarb and metaflumizone. Indoxacarb and metaflumizone belong to the sodium channel blocking insecticides (SCBIs), which have a commom action target site on sodium channels, although chemical structures of them were quite different. Both of them have a good insecticidal potency and excellent mammalian and natural enemies safety. In the present study, cloning and sequencing of the sodium channel were performed in susceptible and resistant strains, and two amino acid substitutions (F1868Y and V1871I) were identified in resistant populations. Then we demonstrated the association between mutations and SCBIs resistance in P. xylostella, using laboratory maintained strains and field-collected populations. The findings in this stduy are very important for further understanding the action mechanisms and target-site resistance mechanisms of SCBIs.1. Cross-resistance and mechanisms of resistance to indoxacarb and metaflumizone in the BY-12 population of P. xylostellaThe field BY-12 population, collected from Baiyun (Guangzhou, China) has developed high levels of resistance to indoxacarb and metaflumizone (728- and 70-fold, respectively). Indoxacarb has been widely used to control P. xylostella for a long time, while use of metaflumizone is very limited in southern china, suggesting that selection of indoxacarb in the field may confer cross-resistance to metaflumizone in the BY-12 population. In the BY-12 population, three inhibitors (PBO, DEM, DEF) had 1.3-to 2.5-fold synergism on metaflumizone. Therefore, we speculated that the cross-resistance between indoxacarb and metaflumizone was caused by the target site mutations. The cDNA sequences encoding the sodium channel gene of P. xylostella (PxNav) were cloned, sequenced and compared between the BY-12 population and a susceptible reference strain (Roth). Two mutations (F1868Y and V1871I) in the segement S6 of domain IV were identified in BY12 population, with 0.1 and 0.425 mutant frequencies, respectively. F1868 and V1871 are highly conserved in insect sodium channel gene, suggesting that these mutations may be associated with resistance. In addition, F1868Y mutation was not detected in the BY-12 subpopulation, which was maintained without any insecticide selection for 24 generations. Meanwhile, resistance to indoxacarb and metaflumizone decreased respectively to 45-fold and 3-fold. The results not only showed F1868Y and V1871I mutations were associated with the cross-resistance between indoxacarb and metaflumizone, but also that metabolic detoxification was involved in indoxacarb resistance in the BY-12 strain.2. Cross-resistance and mechanisms of resistance to indoxacarb and metaflumizone in the SY-14 population of P. xylostellaThe field SY-14 population, sampled from Sanya (Hainan, China) has developed high level resistance to indoxacarb (825-fold), but only 6-fold resistance to metaflumizone. In this population, the mutation frequencies of F1868Y and V1871I were 0.017 and 0.183, and the synergistic effects of DEF and PBO were 9.8-fold and 3.9-fold. The results showed that both metabolic detoxification and sodium channel gene mutation were involved in the indoxacarb resistance in the SY-14 population, furthermore, the contribution of metabolic detoxification was higher than the target-site mutations.3. Frequency detection of F1868Y and V1871I in field populations of P. xylostellaIn order to investigate the association between the two point mutations of PxNav and SCBIs resistance, we detected the mutation frequencies of F1868Y and V1871I in Roth, SZ strains and several field populations collected during 2012-2014. In the strains of Roth and SZ, the two mutations were not detected. In the 12 field populations, V18711 was found in seven populations, with frequencies from 0.022 to 0.133, while F1868Y was only detected in BY-13 population with a 0.033 mutation frequency. Overall, the frequencies of F1868Y and V18711 mutations correlate with resistance levels. In addition, F1868Y and V1871I did not co-exist in a single larvae, the mutually exclusive genotype may be associated with fitness costs.