Characteristics And Mechanisms Of Resistance To Spinosad And Chlorantraniliprole In The Diamondback Moth,Plutella Xylostella

The diamondback moth, Plutella xylostella (L.)(Lepidoptera:Plutellidae), is one of the most damaging, cosmopolitan economic pests of cruciferous vegetables, especially in the tropical and subtropical areas. A conservative estimate of total costs associated with its management is about US$4-5billion every year. Its short generation time, high fecundity, and extensive selection pressure in the field, have resulted in rapid evolution of resistance to various classes of insecticides (including84kinds of active ingredients). P. xylostella has become one of the most difficult pests to control in cruciferous vegetables.Spinosad is the first commercialized member of spinosyn class of insecticides, which primarily attacks the nicotinic acetylcholine receptor. Chlorantraniliprole is the first commercial insecticide from a novel class of chemistry, the anthranilic diamides, which selectively binds to insect ryanodine receptors (RyRs). Both insecticides have high insecticidal potency and good mammalian safety. In the present study, we investigated the characteristics (including resistance selection, stability, cross-resistance and mode of inheritance) and mechanisms for resistance to spinosad and chlorantraniliprole in P. xylostella. The information from our research will be valuable in understanding resistance mechanisms at molecular level and in developing rational resistance management strategies.1. Characterization of spinosad resistance in the SZ-Spin83strain of P. xylostellaThe SZ-Spin83strain developed4,000-fold and10,000-fold resistance to spinosad under laboratory selection when compared with SZ (the progenitor strain of SZ-Spin83) and Roth (the susceptible strain) respectively. With or without continuous selection by spinosad, resistance in SZ-Spin83was stable with no significant change. Compared with the SZ strain, the SZ-Spin83strain showed high levels of cross-resistance to abamectin (468-fold) and spinetoram (2396-fold), no obvious cross-resistance to indoxacarb, beta-cypermethrin, fipronil, chlorfenapyr, cartap, chlorfluazuron, diafenthiuron, tebufenozide, metaflumizone and chlorantraniliprole. Inheritance analysis results showed that spinosad resistance in SZ-Spin83strain of P. xylostella was autosomal, co-dominant, and polygenic.2. Mechanisms of spinosad resistance in the SZ-Spin83strain of P. xylostellaThe synergistic effects of PBO, DEM and DEF on spinosad were determined in the Roth, SZ and SZ-Spin83strains, and the results showed that the three inhibitors did not significantly synergize spinosad against the three strains (SR<2-fold). In order to evaluate the role of metabolic detoxification mechanisms in spinosad resistance, the activities of P450monooxygenases, esterases and glutathione S-transferases were measured in the three strains. Metabolic enzyme activities in SZ-Spin83were0.68～1.81-fold compared with the Roth strain, and there was no significant difference in activities between SZ-Spin83and SZ strains. Enzyme activity results were consistent with those of the synergism experiments, supporting that metabolic detoxification was not involved in spinosad resistance in SZ-Spin83, and taget-site resistance may be the the major resistance mechanism. To investigate possible target site resistance mechanisms, a nAChR subunit gene (Pxa2) was cloned and characterized from SZ-Spin83and Roth strains. There was no conservative difference at amino acid level among the Roth, SZ and SZ-Spin83strains. Pxa2mRNA expression, quantified by real-time PCR, was no significantly different between SZ-Spin83and SZ strains. Recent studies reported that Pxa6gene mutation was associated with spinosad resistance in a strain of P. xylostella. To determine if SZ-Spin83resistance was due to the same mutation,55from Roth and58from SZ-Spin83of Pxa.6transcripts were sequenced and analyzed. Six isoforms of Pxa6were detected, three of which are novel isoforms. However, our results showed no evidence of a selective sweep associated with the Pxa6in the resistant SZ-Spin83strain, and the mRNA expression levels were similar among the resistant and susceptible strains. Our work suggested neither Pxa2nor Pxa6was associated with spinosad resistance in SZ-Spin83. It is suggested that other nAChR subunits or other receptor genes other than nAChR could be responsible for spinosad resistance in SZ-Spin83.3. Baseline susceptibility and resistance monitoring of P. xylostella to chlorantraniliproleThe susceptibility of16field populations and seven laboratory maintained strains of P. xylostella to chlorantraniliprole were determined through leaf dip bioassay during2008-2009. The susceptibility variation among16field populations was low (5-fold), with median lethal concentrations (LC50values) varying from0.221to1.104mg/liter. However, wider ranges of variation in LC50s (10-fold) were observed among seven laboratory strains. A discriminating concentration (15mg/liter) was calibrated from pooled toxicological data of the16field populations, seven laboratory strains and five field populations showed an average mortality of99.75%(from98to100%). The results indicated field P. xylostella populations were sensitive to this chemical, will also be useful in resistance monitoring and early warning.20field populations of P. xylostella sampled in2010-2011from China were tested with laboratory bioassays to determine if susceptibility to chlorantraniliprole had changed in the field. The LC50s was about three fold (from0.226to0.71mg/liter) among the14field populations from northern China, but reached as high as770-fold (from0.343to265.2mg/liter) among the six field populations from southern China (Guangdong Province). Among the six populations, very high levels of resistance were detected in Zhuhai (ZH)(150-fold) and Zengcheng (ZC)(2140-fold). The present work suggests that we should rational use chlorantraniliprole, inorder to extend the useful life of this compound. Meanwhile, we urge to stop use of chlorantraniliprole in somewhere if high level of resistance had evolved in field.4. Characterization of field-evolved resistance to chlorantraniliprole in P. xylostellaThree field populations of P. xylostella collected from southern China in2011showed parallel resistance levels to chlorantraniliprole (from18-to1150-fold) and flubendiamide (from15-fold to800-fold), indicating strong cross-resistance between these two diamide insecticides. In the absence of selection pressure, resistance to chlorantraniliprole in ZC population declined from2040-fold (Gi) to25-fold (G7). Genetic analysis showed that chlorantraniliprole resistance in the ZC population was autosomal and incompletely recessive. The ZC-R strain (derived from ZC) had670-fold resistance to chlorantraniliprole. Synergist bioassays performed on ZC-R showed chlorantraniliprole toxicity was synergized by PBO, DEM and DEF at low levels. This work showed metabolic detoxification was involved in chlorantraniliprole resistance in the ZC-R strain to some extent, and target-site resistance may be the major mechanism. 5. Relationship between variations of the ryanodine receptor of P. xylostella and chlorantraniliprole resistanceThe full-length cDNA of a ryanodine receptor gene (PxRyR) was cloned and characterized from P. xylostella by RNA-Seq, RT-PCR and RACE technologies. The cDNAs of PxRyR contain a15,495-bp open reading frame,267-bp5’untranslated region (UTR) and a3’-UTR of351-bp. The predicted mature protein consists of5164amino acids with a predicted molecular weight of583.7-kDa. PxRyR shares common structural features with known RyRs:the well-conserved COOH-terminal domain, which forms a functional Ca2+channel, and a large hydrophilic NH2-terminal domain. PxRyR shows a high level of amino acid sequence identity (78-80%) to the insect RyR isoforms. Ten deletion polymorphism sites were detected in PxRyR cDNAs, suggesting a single PxRyR can produce many polymorphic transcripts. Although the highest mRNA expression level was observed in larva and the lowest in pupa, there was a relatively stable expression during the developmental period from egg to adult. The relative mRNA expression levels of PxRyR were similar among the head, thorax, and abdomen of the fourth-instar larva body.We used the A/C polymorphism at the site13,349bp as a molecular marker of PxRyR cDNA to test the linkage between PxRyR locus and chlorantraniliprole resistance in the ZC-R strain. Backcross analysis results showed that chlorantraniliprole resistance was genetically linked with PxRyR in ZC-R. We sequenced a cDNA fragment encoding1691amino acids in the COOH-terminal domain of PxRyR. Two point mutations (I4790K, G4946E) were existed in resistant ZC-R strain, but only G4946E was associated with resistance to some extent. PxRyR mRNA expression levels in the resistant ZC-R strain, quantified by real-time PCR, was reduced to41～46%of the Roth and SZ strains. It was concluded that chlorantraniliprole resistance was linked with the PxRyR locus, and both amino acid mutations and reduced mRNA expression of PxRyR could be involved in resistance.