Resistance Mechanisms To Spinosad In Diamondback Moth, Plutella Xylostella (L.)

The diamondback moth(DBM),Plutella xylostella L.(Lepidoptera:Plutellidae), mainly damages cruciferous plants,and is one of the most destructive pests of cruciferous vegetables.It causes enormous losses to vegetables production every year,requiring globally US $1.0 billion in estimated annual management costs.Moreover,it is also one of the pests which can develop most serious resistance to insecticides,and has developed significant resistance to more than 50 kinds of insecticides,including almost every insecticide applied in field.Spinosad is one of the novel macrolides with perfect efficacy against DBM resistant to conventional insecticides,because of its unique mode of action.In this paper,the cross resistance pattern and resistance mechanisms to spinosad in DBM were investigated based on a spinosad-selected strain of DBM,in order to provide essential scientific basis for designing resistance management strategies.1 Selection for resistance to spinosad and cross resistance pattern of spinosad resistance in DBMThe SZ-Spin56 strain was selected from a field-collected strain(SZ) with spinosad in laboratory.After 30 generations of selection,resistance of the SZ-Spin56 strain to spinosad increased to 170.3-fold compared with a susceptible strain(ROTH),and 65.9-fold compared with the SZ strain.However,resistance of the SZ-Spin56 strain to abamectin, indoxacarb,beta-cypermethrin,fipronil,cartap,chlorfenapyr,metaflumizone,tebufenozide, chlorfluazuron did not increased compared with the SZ strain,these results indicated that there was no cross resistance between spinosad and these insecticides.It suggests that rational rotation or mixture of these insecticides with spinosad in field control of DBM could slow down resistance development to spinosad and thus prolong application longevity of spinosad.2 Inheritance mode of resistance to spinosad in DBMThe inheritance mode of resistance to spinosad in DBM was evaluated by using the methods of reciprocal crosses and backcross between the susceptible strain ROTH and spinosad-resistant strain SZ-Spin56.The progenies of reciprocal crosses showed 11.1-fold (F₁) and 10.5-fold(F₁′) resistance to spinosad compared with ROTH strain,respectively, and there was no significant difference between the LC₅₀ values of F₁ and F₁′,indicating that the resistance of DBM to spinosad was autosomal.The dominant degrees of F₁ and F₁′were -0.06 and -0.08,respectively,indicating that the resistance of DBM to spinosad was semidominant.In the LD-P line for spinosad to the progenies of backcross between F₁ and SZ-Spin56,there was no distinct plateau corresponding to 50%mortality,and theχ² test also confirmed that the resistance of DBM to spinosad was probably polygenic.3 Biochemical characterization of spinosad resistance in DBMThe role of detoxification enzymes in spinosad resistance of the SZ-Spin56 strain was preliminarily studied by using synergist bioassay and detoxification enzyme assay.The results of synergist bioassay showed that enzyme inhibitors,DEF,DEM and PBO had no significant synergism to spinosad in the susceptible strain ROTH,control strain SZ and spinosad-resistant strain SZ-Spin56.The results of detoxification enzymes assay showed that the activities of MFO(MROD,ECOD) in SZ strain and SZ-Spin56 strain were higher than that in ROTH strain,but there was no significant difference between SZ strain and SZ-Spin56 strain.There was no significant difference in the activities of GST with substrate CDNB among the three strains,on the other hand,the activities of GST with substrate DCNB in SZ strain and SZ-Spin56 strain were higher than that in ROTH strain,but there was no significant difference between SZ strain and SZ-Spin56 strain.The activities of esterases in SZ strain and SZ-Spin56 strain were higher than that in ROTH strain,but there was no significant difference between SZ strain and SZ-Spin56 strain.These results indicated that spinosad resistance observed in the SZ-Spin56 strain was possibly attributed to decreased target site sensitivity,and the detoxification metabolic enzymes may have little contribution.4 Cloning and sequence analysis of nicotinic acetylcholine receptor genes from Plutella xylostellaNicotinic acetylcholine receptors(nAChR.) mediate the fast action of excitatory neurotransmitter at cholinergic synapses in the central nervous system of insects and are also the target site for neonicotinoids and spinosad.By using RT-PCR and RACE technique, the full-length cDNA of a novel nAChRαsubunit(Pxα8) was cloned from Plutella xylostella(GenBank accession number:EU914853).Sequence analysis showed that the full-length cDNA was 1744bp in length and contained an open reading frame(ORF) of 1602bp which encoded 534 amino acid residues.The deduced amino acid sequence of Pxα8 shared typical features of nAChRαsubunits and showed 77%-96%amino acid similarity with insect nAChRα8 subunit orthologues.Interestingly,it also showed 76%amino acid similarity with the nAChRβ2 subunit of Drosophila melanogaster.There were many single nucleotide polymorphism(SNP) sites in the ORF of Pxα8,some of which resulted in amino acid substitution.There are more SNPs in female 4th-instar larvae than that in male 4th-instar larvae,and the SNP sites were different between the female and male 4th-instar larvae.There were more amino acid polymorphism sites in spinosad-resistant strain SZ-Spin56 than that in SZ strain,and the amino acid polymorphism sites were different between SZ-Spin56 and SZ strain.Semi-quantitative RT-PCR analysis indicated that the mRNA expression level of Pxα8 was higher at adult stage than at pupal and 4th-instar larval stage.These findings provide important basis for further study on diversity of nAChR subunits and mechanism of target resistance to spinosad in P.xylostella.