Utilization Of Near-Isogenic Lines To Identify Effects Of The Ryanodine Receptor Point Mutations On Diamide Resistance In Plutella Xylostella

Diamondback moth,Plutella xylostella（Lepidopteran Plutellidae）,is one of the most damaging economic pest of cruciferous in the world with more than 40 hosts.Diamide insecticides are a new type of chemical with fast insecticidal speed and target selectivity.They can effectively control Lepidopteran pests such as Plutella xylostella.The global sales of the diamides insecticides were more than 2 billion dollars.With properties of short generation cycle,high fertility,overlapping generation and the unreasonable use of pesticides in the field,the diamondback moth has developed different levels of resistance to the insecticides used to managment it.With 2 to 3 years application of chlorantriniliprole,high resistant populations of P.xylostella were detected in the field.The useful life of diamides and the production of cruciferous crops were seriously threated by resistance development of P.xylostella.The current research indicates that specific mutations in the ryanodine receptor（Ry R）of the P.xylostella,can confer resistance to diamide insecticides.In this paper,the 4946E,4790M,and 4790K alleles of Px Ry R were introgressed into the genetic background of a susceptible reference strain,IPP-S,using molecular marker-assisted near-isogenic line selection technology.The contribution these three introgressed alleles had on the diamide resistance phenotype was determined,then inheritance and genetic linkage between flubendiamide and resistance was tested in each strain.Moreover,we compared a series of fitness traits between the three introgression strains and susceptible reference.This study provides systemic information about the extent of diamide resistance mediated by Ry R mutation in P.xylostella and its associated genetic traits.Our findings can be used to guide diamide resistance management practice for this species and other arthropods pests.1.Construction of 4946E,4790M and 4790K near-isogenic lines and determination of its resistance phenotypeUsing the diamide resistant populations BY-19 and SH-19 collected in the field as male parents,the Px Ry R G4946E,I4790M and I4790K mutant alleles were intrigressed into the susceptible strain IPP through the multi-generation backcross assisted by molecular markers.Three homozygous strains containing single point mutation were finally successfully obtained,and named as 4946E,4790M and 4790K respectively.each introgressed strain（4946E,4790M and 4790K）was homozygous for the corresponding mutant allele and shared about 94%（15/16）of its genetic background with that of the recipient IPP-S strain.The toxicity test results showed that mutations alone can result in moderate to high levels of resistance to five commercial diamides（flubendiamide,chlorantraniliprole,cyantraniliprole,tetraniliprole,and cyclaniliprole）,and the resistance intensity mediated by the three mutations was hierachial in order of 4790K（1199-to>2778-fold）>4946E（39-to 739-fold）>4790M（16-to 57-fold）.This study shows that the resistant homozygous strains obtained by gene introgression technology can effectively assess the contribution of a single Px Ry R point mutation to the diamide resistance.The results provide important information for early warning of cross-resistance among diamides,and could guide resistance management practices for diamides in P.xylostella.2.The genetic traits of resistance to flubendiamide in 4946E,4790M and 4790K near-isogenic linesThe susceptible IPP-S strain and each genetically pure strains（4946E,4790M and4790K）were mass crossed and the toxicities of flubendiamide against their F₁progeny from reciprocal crosses were tested.The LC₅₀s of F₁progeny from reciprocal crosses had no significant difference,with overlapping 95%fiducial limits,and the dominance values（D）were-0.73、-0.47 and<-0.85 for flubendiamide,respectively.These data demonstrate autosomally and incompletely recessive mode of inheritance for flubendiamide resistance in the 4946E,4790M and 4790K strains,respectively.For genetic likage analysis of the mutant allele and flubendiamide resistance,IPP-S adults were firstly reciprocally mass-crossed with moths from the mutation introgressed strain to produce F₁progeny.Male moths from the pooled F₁progeny were mass-backcrossed to the female moths from the introgression strain to generate backcross progeny.When treated by the diagnostic concentration of flubendiamide,52.3%,49.2%and 49.7%larvae survived（be consistent with the theoretical ratio if resistance is monogenic）after 48 h exposure in three BC groups.The genotype of the Px Ry R locus（4790 or 4946）was determined by direct sequencing technology,and it was found that all the surviving individuals were homozygous for carrying the corresponding mutant gene,while the untreated individuals were basically separated as rr:rs=1:1.The results showed that the introgressed Px Ry R resistance genes are closely linked with flubendiamide resistance of 4946E,4790M and4790K near-isogenic lines.3.Relative fitness comparison of 4946E,4790M and 4790K near-isogenic lines of P. xylostellaTo determine the potential fitness cost,life tables of the susceptible IPP-S and the introgressed resistant strains（4946E,4790M and 4790K）were constructed.The main parameters,including the larval development time,pupal rate,pupal stage,pupal weight,pupal emergence,the proportion of females and the rate of egg hatching were recorded and calculated.According to the net replacement rate（R₀）of the populations,the relative fitness of the 4946E,4790M and 4790K strains were estimated to 0.77,0.93 and 0.92,respectively,compared with the background strain IPP-S（R₀=1.00）.This indicates that there are relatively higher fitness costs associated with diamide resistance in 4946E than4790M and 4790K strains of P.xylostella under laboratory conditions.Furthermore,we speculate that 4790K mutation,with strong contribution to diamide resistance and lower fitness cost,will increase prevalence in the field.