Detection Of Frequencies Of Target-site Resistance Alleles In Field Populations Of Plutella Xylostella And Inheritance Of Target-site Resistance To SCBIs

The diamondback moth,Plutella xylostella(L.)is a major lepidopteran pest to cruciferous vegetables worldwide.The biological characteristics such as strong reproductive capacity,short generation time and multiple overlapping generations lead to rapid evolution of resistance to various classes of insecticides.So far,the diamondback moth has developed resistance to at least 93 kinds of insecticides.The resistance development of P.xylostella is threatening the sustainable production of cruciferous vegetables.A number of research shows that reduction of target susceptibility is one of the major mechanisms responsible for high levels of resistance,and mutations in several target genes have been associated with insecticide resistance in P.xylostella.In this study,we detected the mutant allele frequencies of a number of mutations related with resistance in field populations of P.xylostella by sequencing of the target DNA fragments,and correlations between mutant allele frequencies and resistance levels were assessed.In addition,we studied inheritance modes of resistance to metaflumizone and indoxacarb in the KS-1845Y strain(homozygous for F1845Y mutation of the sodium channel gene)and the KS-1848I strain(homozygous for the V18481 mutation)of P.xylostella.Our results will provide an important basis for developing resistance management strategies of P.xylostella in China.1.Monitoring of insecticide resistance levels in field populations of P.xylostellaResistance levels to 7 insecticides were measured by leaf-dipping method in the field populations collected from East China and Guangdong province during 2014-2015.The results showed that varying levels of resistance to individual insecticides were developed in the field populations tested.Field populations from Guangdong province showed high levels of resistance to beta-cypermethrin,indoxacarb,chlorantraniliprole and chlorpyrifos,and medium levels of resistance to metaflumizone,abamectin and spinosad.Except for Jinan population developed high-level resistance to beta-cypermethrin(about 130-fold),the other populations collected during 2014-2015 from four regions of East China,showed medium to low levels of resistance to indoxacarb,metaflumizone,chlorantraniliprole,abamectin and chlorpyrifos.2.Detection of target-site resistance allele frequencies in field populations of P.xylostellaBesides measuring resistance levels,allele frequencies associated with target site resistance were also detected in the above field populations of P.xylostella.The allele frequencies of the sodium channel gene containing M9181,T9291 or L1014F substitutions were 0-11.6%,86.6%-100%and 96.6%-100%,respectively.No obvious relationship was observed between these mutation frequencies and pyrethroid resistance levels.We speculated that there are other resistance mechanisms conferring pyrethoid resistance in these field populations.The mutation frequencies of another two residues in the sodium channel,F1845Y and V1848I were 0-23.3%and 0-40%,respectively,and their pooled frequencies had a significant correlation with resistance levels to indoxacarb(R2=0.822)and metaflumizone(R2=0.887).It suggested that the target-site resistance mediated by F1845Y or V1848I mutations was a major resistance mechanism to SCBIs insecticides at least in the P.xylostella field populations tested.The mutation frequencies of the ryanodine receptor gene with E1338D or I4790M substitutions were 0-10%and 0-8.3%,resepetively,and most of them were present as heterozygotes.By contrast,the frequency of G4946E was from 21.6%to 90.3%,with a significant correlation with resistance levels to chlorantraniliprole(R2=0.75).We therefore concluded that G4946E muations could be involved in high levels of resistance to chlorantraniliprole.The mutation frequency of A309V of the glutamate-gated chloride channel gene was 0-3.3%and was only detected in Kunshan population.Mis-splicing of a6 subunit of the nicotinic acetylcholine receptor was not detected in these field populations,other resistance mechanisms are supposed to confer resistance to spinosad.The mutation frequencies of A298S and G324A of the acetylcholinesterase-1 gene were 41.6-75.1%and 41.6-75.1%,respectively,which had a significant correlation with resistance levels to chlorpyrifos(R2=0.863 for A298S,R2=0.847 for G324A).It suggested that the A298S and G324A mutations were the main resistance mechanism to chlorpyrifos in the field populations tested.3.Inheritance of resistance to SCBIs in two resistant strains of P.xylostellaPrevious studies have demonstrated that two point mutations(F1845Y and V1848I)in the sixth segment of domain IV of the sodium channel gene are associated with resistance to indoxacarb and metaflumizone in P.xylostella.In order to confirm contributions to resistance and inheritance modes of the two point mutations,the KS-1845Y strain(homozygous for F1845Y mutation)and the KS-18481 strain(homozygous for the V1848I mutation)were isolated with single-pair family selection from a field population collected from Kunshan,Jiangsu province in 2015.The KS-1845Y and KS-1848I strains were respectively crossed with a susceptible strain(SZ),and the three parent strains and their F:progeny were bioassayed to determine their susceptibility to indoxacarb and metaflumizone.The KS-1845Y and KS-18481 strains exibited similar levels of autosomally inherited resistance to indoxacarb(381-and 336-fold)and metaflumizone(736-and 674-fold).The two strains showed semi-domiant to incompletely domiant resistance to indoxacarb and metaflumizone,but the KS-1845Y strain had higher dominance levels of resistance to the two insecticides than the KS-1848I strain.Differential interaction between the two SCBI insecticides and the two mutant sodium channel proteins may result in different degrees of the dominance levels.Under selection pressure posed by indoxacarb or metaflumizone,resistance evolution in field populations of P.xylostella could favor the F1845Y mutation with relatively higher dominance.