Study On Resistance Inheritance And Resistance Mechanism Of Tetranychus Urticae To Cyetpyrafen

The two-spotted spider mite（Tetranychus urticae）has been recognized as an important pest in agriculture and forestry and chemical control is still considered as the most effective strategy against T.urticae in agriculture.However,the unreasonable and inappropriate application of pesticides and the characteristics of short reproductive cycles and abundant generation of T.urticae have led to the more serious problem of resistance in these mites.At present,there is a dwindling number of pesticides that has acaricidal activity against T.urticae.Cyetpyrafen is a novel acrylonitrile-like acaricide independently developed in China and officially launched in 2017.The subunits of mitochondrial complex II,succinate dehydrogenase（SDH）is the action site of cyetpyrafen.Thus,it is a SDHI acaricide and also belongs to mitochondrial electron transport chain inhibitors（METI）.In addition,cyetpyrafen has the broad spectrum of highly acaricidal activities against all developmental stages of mites with quick and long-lasting effect.Meanwhile,cyetpyrafen has a completely new chemical structure.Thus,it has no cross-resistance with non-SDHI acaricide that are commonly used in the field and does not display harmful effects against non-target organisms.The cyetpyrafen shows promising properties for applications in the control of T.urticae.Therefore,studies on T.urticae resistance to cyetpyrafen are of great significance in the rational use of pesticides,management of field resistance,and extending the lifetime of pesticides.In the current study,two susceptible strains（Tu-YN,Tu-DG）and one cyetpyrafen resistant strain（Cyet-R）were used to clarify the inherited forms,fitness,and mechanisms of cyetpyrafen resistance.The results were aimed to provide reference evidence for the scientifically appropriate use of cyetpyrafen.Specific results are as follows:1.The resistance levels and inherited forms of Cyet-R to cyetpyrafenThe levels of resistance in three strains were examined through leaf-disc spraying methods.The results showed that LC₅₀（lethal concentration 50%）estimated for the Cyet-R strain was larger than 10000 mg/L,2000-fold higher than that of the Tu-YN strain,reaching extreme high resistance level.Meanwhile,the Cyet-R strain also displayed extreme high resistance levels against the other two SDHI acaricides,with LC₅₀larger than 10000 mg/L and 2600-fold higher resistance levels to cyenopyrafen,and LC₅₀at2818.99mg/L and 190-fold higher resistance levels to cyflumetofen.The inherited forms of cyetpyrafen resistance were examined through reciprocal cross and backcrossing experiments,combining with bioassay.The results showed that the LC₅₀for generation F_1RS and F_1SRwere 3126.30 mg/L and 2743.97 mg/L,respectively,with overlapping 95%confidence interval.There was no significant difference between F_1RS and F_1SR through hypothesis testing and regression analysis.In addition,the dose-response curve of F_1RS or F_1SR was almost perfectly with the other.These results suggested that inherited forms of cyetpyrafen resistance was nuclear inheritance.The degrees of dominance of F_1RS or F_1SR ranged between 0 and 1 and both dose-response curves were closer to the resistant parent,indicating that the inheritance of cyetpyrafen resistance was incompletely dominant.The real mortality rates of backcrossing strains BC₁and BC’₁was different from the expected mortality rates based on assumed a monogenic inheritance model.Moreover,the actual chi-square value was significant different from the expected value based on chi-square test.Therefore,resistance to cyetpyrafen is governed by polygenes.2.The population fitness of Cyet-RThe two-sex life table was analyzed with TWOSEX-MSChart software.The results showed that the Cyet-R strain had shortened maturation-stage for adult mite.The time for nymphs was significant shorter than that of Tu-YN strain and time for eggs and larvae was significant shorter than that of Tu-DG strain.The time for oviposition period and female adults was significant prolonged in Cyet-R strain.In addition,no significant differences were found for the intrinsic increase rate,finite rate of increase,net reproductive rate and mean generation time（T）of the three strains.Nevertheless,the female fecundity of Cyet-R strain was significant higher with peak spawning twice per day.Thus,the cyetpyrafen resistant strain have no significant fitness cost.3.The biochemical mechanism of T.urticae resistant to cyetpyrafenAfter separate pre-exposure to PBO,DEM,and DEF synergists for 24 h,the female adults of three strains were evaluated by using bioassay with cyetpyrafen.The synergistic ratios of PBO to cyetpyrafen was 1.7 and 5.1 in Tu-YN and Tu-DG strains,respectively.The synergistic ratios of DEM and DEF to cyetpyrafen was separately 2.1 and 1.5 in Tu-DG strains.Moreover,the three synergists had no effect on the sensitivity of Cyet-R strain to cyetpyrafen.Besides,the specific activity of esterases（CCEs）in Cyet-R strain was separately 1.10 times higher than that in Tu-YN and Tu-DG through detoxification enzyme activity assays,whereas the specific activities of P450s and GSTs showed no significant differences among the three strains.Taken together,those results suggested that P450s and CCEs might participate in the cyetpyrafen metabolism in T.urticae.Nevertheless,metabolic detoxification had a lower contribution to cyetpyrafen resistance on the whole.4.Differentially expressed genes associated with detoxification enzymesTranscriptome analysis showed that 313 DEGs were identified in Cyet-R strain（183upregulated,130 downregulated）,including six CCEs and three P450s genes.RT-q PCR validation confirmed the upregulation of five CCEs genes（Tu CCE01,Tu CCE21,Tu CCE47,Tu CCE48,and Tu CCE71）and two P450s genes（CYP389C12 and CYP392D3）in Cyet-R strain.In addition,the expression levels of Tu CCE01,Tu CCE47,Tu CCE48,and CYP392D3 had higher fold changes,suggesting their potential roles in cyetpyrafen resistance.5.Targets for cyetpyrafen resistance in T.urticaeThe full-length of four subunits of SDH（Tu Sdh A,Tu Sdh B,Tu Sdh C,and Tu Sdh D）were cloned from the three strains and sequenced.Then,blast analysis found the site mutation of Tu Sdh B(Sdh B^I260V)in Cyet-R strain.Near-isogenic line Sdh B^I260V strain was constructed and showed about 462-fold resistance levels to cyenopyrafen.Although the resistance levels were lower than the Cyet-R parent,the mutant strain still displayed extreme high resistance levels.This mutation reduced the ability of cyenopyrafen binding to target sites through examining the inhibitory effects of cyenopyrafen to SDH.These results indicated that Sdh B^I260V mutation of target gene presented an important contribution for cyenopyrafen resistance.Taken together,the cyetpyrafen resistant strain of T.urticae has developed obvious cross-resistance to cyenopyrafen and cyflumetofen.The inherited form of cyetpyrafen resistance is nuclear inheritance and controlled by polygenes with incomplete dominance.The cyetpyrafen resistant strain Cyet-R also showed a fitness advantage.The site mutation of Tu Sdh B(Sdh B^I260V)is supposed to be an important cause of cyetpyrafen resistance mechanism for Cyet-R strains.Meanwhile,metabolic detoxification had a lower contribution to cyetpyrafen resistance.