Functional And Population Fitness Studies On Target Mutations Of Cyetpyrafen Resistant Tetranychus Urticae

Tetranychus urticae（Koch）is a significant and serious agricultural mite.Resistance develops quickly due to its short life cycle and high fecundity.Target mutation is generally regarded as an important factor leading to high resistance in the study of resistance mechanisms,and many target mutations have been shown to be associated with the formation of resistance to acaricides.Cyetpyrafen is an acaricide that belongs to the mitochondria complex II inhibitor（SDHI）class.Previous research in our laboratory on the resistance mechanism of T.urticae target identified the mutations I260V in B subunit and R119C in D subunit from the SDH of cyetpyrafen-resistant population.To evaluate the relative contribution in cyetpyrafen mite resistance and fitness effects of different SDH mutations in T.urticae.In this study,the near-isogenic line with SDH mutation of T.urticae was constructed,and the mutation was introduced into the genetic background of susceptible lines.Bioassays and in vitro inhibition of SDH activity were used to assess the role of different SDH mutations in cyetpyrafen mite resistance,and the fitness costs of SDH mutations were assessed by constructing an age-stage,two-sex life table.The following are specific research findings:1.Construction and verification of near-isogenic lines with SDH mutation in T.urticaeUsing introgression method based on trait selection,Cyet-R（RR>2400）was chosen as the donor parent and Tu-YN as the recipient parent.Three near-isogenic lines with SDH mutation were obtained after seven generations of backcrosses.Tu-YN^I260V was charactered with Sdh B（I260V）mutation,Tu-YN^R119C was charactered with Sdh D（R119C）mutation,and Tu-YN^I260V+R119C was mutated with both Sdh B（I260V）and Sdh D（R119C）.ISSR molecular markers were used to examine the genetic background of the isogenic lines,and the results revealed that the genetic consistency between the three near-isogenic lines and the susceptible strain Tu-YN was greater than 0.92,and the genetic distance was less than 0.09.According to the UPGMA cluster analysis,the near-isogenic line with SDH mutation was closely related to the parent Tu-YN.2.The role of SDH mutation in T.urticae mite in cyetpyrafen resistanceBioassays of cyetpyrafen resistant Cyet-R strain,susceptible Tu-YN strain and three near-isogenic line with SDH mutation were performed by spraying method.The LC₅₀values for cyetpyrafen against Tu-YN^I260V and Tu-YN^R119Cwere 1375.42 mg/L and 30.20mg/L,respectively.Tu-YN^I260V and Tu-YN^R119Cshowed 341.30 and 7.49 fold resistance to cyetpyrafen,respectively,when compared to the susceptible strain Tu-YN.The LC₅₀was greater than 10000 mg/L when both Sdh B（I260V）and Sdh D（R119C）mutations were present.These results showed that the resistance level of Tu-YN^I260V+R119C was as high as that of Cyet-R,with a resistance ratio greater than 2481.39.These results indicated that I260V mutation on Sdh B of T.urticae resulted in a high level of resistance to cyetpyrafen,and the presence of both Sdh B（I260V）and Sdh D（R119C）mutation resulted in a very high level of resistance to cyetpyrafen comparable to that of resistant strain Cyet-R.The enzyme kinetic parameters of the SDH protein of T.urticae were determined,and these results showed that the Km values of the SDH proteins of the susceptible Tu-YN strain and the three near-isogenic lines with SDH mutation were nearly identical.However,the Vmax value of SDH protein decreased significantly when the mite harbored Sdh B（I260V）and Sdh D（R119C）alone or in combination,compared to the susceptible Tu-YN strain.The Vmax value of SDH protein was only 7.53 nmol/min/mg pro when both Sdh B（I260V）and Sdh D（R119C）mutations were present,which was less than one-third of the Vmax value（25.40 nmol/min/mg pro）of the susceptible Tu-YN strain.Thees results of the in vitro inhibition of SDH activity of T.urticae demonstrated that the inhibitor cyetpyrafen had a non-competitive effect because the SDH protein’s in vitro Michaelis constant Km remained unaltered after it was added and the maximal response rate Vmax dropped.In addition,the inhibitory constant Ki of cyetpyrafen on SDH of T.urticae rose dramatically when Sdh B（I260V）and Sdh D（R119C）mutations were present,either separately or combinely.The inhibitory constant Ki of cyetpyrafen against the SDH of T.urticae was 4969.25 n M,which was 537 times higher than that of the susceptible Tu-YN strain（9.24 n M）,especially when both Sdh B（I260V）and Sdh D（R119C）mutations were present on the SDH.The results were consistent with the bioassay results,indicating that the resistance level was closely related to the target mutation that reduced the inhibition intensity of cyetpyrafen on SDH activity.3.Assessment of fitness effects of SDH mutation in T.urticaeFor the susceptible Tu-YN strain and three near-isogenic lines with SDH mutation,an age-stage,two-sex life table was created in order to assess the fitness impact of the SDH mutation in T.urticae.The results showed that the developmental duration of the isogenic line Tu-YN^I260V+R119C was significantly increased compared with the other three lines.The three near-isogenic line with SDH mutation had decreased fertility,an adult preoviposition period,and an increased proportion of male adult mites when compared to the Tu-YN strain.The intrinsic rate of increase（r）and the finite rate of growth（λ）of the three near-isogenic lines with SDH mutation were much lower than those of the Tu-YN strain,according to a comparison of population parameters.These parameters of net reproductive rate（R₀）of Tu-YN^R119C and Tu-YN^I260V+R119C were significantly lower than that of Tu-YN strain.These results indicated that the mutation of Sdh B（I260V）and Sdh D（R119C）led to the fitness cost of T.urticae,the cost of population fitness was more obvious when combined mutation occurred.