The Molecular Mechanism Of The Selectivity Of Cyflumetofen/AB-1 Between Pest And Predatory Mites Mediated By Target-site Difference

Spider mites,such as Tetranychus cinnabarinus,Tetranychus urticae and Panonychus citri,are polyphagous arthropod herbivores that have a large impact on more than 1,100 plant species,including agriculture,horticulture,and livestock farming.The Integrated Pest Management（IPM）strategy,is a crucially effective and economical approach in agriculture,of which biological and chemical control tactics are combined ideally to improve the long-term stability of agricultural system which preventing pest outbreaks.That is,acaricides are applied to decline the pest mite population efficiently and predator mites control the population of pest mite below the economic threshold continuously.However,most of the exsiting acaricides developed on the basis of toxicity against pest mites rather than molecular target,which show low selecitive toxicity between pest mites and non-target organisms.They kill the predatory mites while controlling the pest mites,breaking the balance of the mite-natural enemy system and easily leading to rampant of pest mites.Interestingly,some recently developed and commercialized acarcides specifically targeting SDH（succinic dehydrogenase,SDH）,including cyflumetofen,cyenopyrafen,pyflubumide and cyetpyrafen,which are highly selective and efficient against the SDH of the spider mites but not against other non-target organisms.However,the molecular mechanism of selectivity of SDHIs remains elusive,which hinders the further development of new selective pesticides.In this study,we systematically investigate the mechanism underlying the selectivity of cyflumetofen/AB-1 between the pest spider mites and predatory mite.The main results were following as below:1.Study on the selective toxicity of cyflumetofen between pest mites and predatory mitesWe analyzed the susceptibility of strains from different geographical origin of the pest mite species T.cinnabarinus and P.citri and the predatory mite N.barkeri to four SDHI acaricides.The LC₅₀ values of cyflumetofen against three pest mite strains Tc-SS,Tc-YN and Pc-CQ were 7.53 mg/L,2.52 mg/L and 5.77 mg/L,respectively,while LC₅₀values against three predatory mite strains Nb-CQ,Nb-BJ and Nb-FJ were all above100,000 mg/L.The selectivity factor（SF）of cyflumetofen for the pest mites over the predatory mite therefore exceeds 13,000 fold.Similarly,for the other three known SDHIs,cyenopyrafen,cyetpyrafen and pyflubumide,the SFs also exceed 11,000,13,000 and 15,000 fold,respectively.These results suggest that four SDHIs might have common properties contributing to their high selectivity between the species tested.Then,we measured the amount of cyflumetofen and AB-1 in the mite body T.cinnabarinus（Tc-SS strain）and N.barkeri（Nb-CQ strain）3 hours after the application of cyflumetofen.The amounts of cyflumetofen were similar between T.cinnabarinus（5.53±0.82μg/100 mg mites）and N.barkeri（5.74±1.06μg/100 mg mites）.In addition,the amount of AB-1 in T.cinnabarinus（0.12±0.06μg/100 mg mites）was lower than the one in N.barkeri（0.19±0.05μg/100 mg mites）,although the difference was not significant.Synergism experiments showed that three synergists（PBO,DEM and DEF）had different effects on T.cinnabarinus and N.barkeri.The synergism rations were 1.42,2.25 and 0.51 on T.cinnabarinus,while the LC_50s of cyflumetofen with three synergists to N.barkeri were still too high,over 100,000 mg/L,to calculated out.These results suggest that the penetration,activation and detoxification are most likely not the key factors that determine the observed selectivity.2.Different effect of cyflumetofen/AB-1 on target SDH of pest and predatory mitesThe IC₅₀ of AB-1 on SQR activity on mitochondrial preparations of the predatory mite N.barkeri（682.26 n M）was 381 fold and 198 fold higher than those determined for T.cinnabarinus（1.79 n M）and P.citri（3.45 n M）,respectively.In T.cinnabarinus（Tc-SS strain）,treatment with 10 mg/L cyflumetofen led to a continuously reduction of the amount of ATP in Tc-SS,and after 3 hours the ATP content was reduced by 66%.In contrast,treatment did not have any effect on ATP content in the cyflumetofen-treated predatory mite（Nb-CQ strain）.In T.cinnabarinus,cyflumetofen led to a continuous increase inhibition rate of respiration,and the relative respiration inhibition rate was 46%at 3 hours.However,the relative respiration inhibition rate was no difference between different treatment within 3 hours.Together,these confirm that the difference in the inhibitory effect of cyflumetofen/AB-1 on the target could lie at the basis of selectivity.3.Study on the selective toxicity action sites mediated by the difference of target enzymes SDH of cyflumetofen/AB-1 between pest and predatory mitesFirst,sequence alignments were performed among species with resolved SDH structures,together with mites of the Tetranychidae and Phytoseiidae families.Although there was overall high conservation among different species,especially in subunit B and subunit C,some variations present could result in species-specificity of the inhibitors.The top docking solution from Tc SDH showed a more negative docking score compared to the one from Nb SDH,-8.5 versus-7.4,suggesting a higher binding affinity of AB-1 with Tc SDH,consistent with the results from the toxicity and biochemical assays.The main difference between the two binding pockets lies in the lid-loop.Both pest mites have a conserved Tyr-Thr motif in the lid-loop,while in both predatory mites and other representative non-target organisms this motif is replaced by a His-X-X motif,which just locates at the feasible interaction area between ligand and amino acid residues from the sequence alignment result above.To validate this model,we switched the lid-loop motifs between Tc SDH and Nb SDH.As expected,the mutant Tc SDH showed an increased docking score compared to WT Tc SDH（-6.8 versus-8.5）,and reversely the mutant Nb SDH showed a reduced docking score compared to WT Nb SDH（-8.8 versus-7.4）,supporting the importance of this motif in cyflumetofen/AB-1selectivity.In order to validate our docking score,we have also rescored our docked poses with the Prime MM-GBSA approach,a widely used method to rank the binding affinity of ligands inside the binding pocket of the receptor.Based on the results,it was observed that WT and mutant Nb SDH showed the binding free energy of-48.606 and-52.930 kcal/mol.In case of WT and mutant Tc SDH,the binding free-energy value was about-58.192 and-56.486 kcal/mol,respectively.Based on the higher binding free values,it was concluded that the pest mites have a higher binding affinity than the predatory mites,which is consistent with the results of our biochemical studies.4.The relationship between the difference of key amino acid motifs and the toxicity selectivity of cyflumetofen/AB-1 was verified by CRISPR/Cas9We introduced the key amino acid motif into the D.melanogaster,and obtained the key amino acid motif characteristics of the HLT-YT strain and the HLT-LT strain simulated pest mites for functional verification.The toxicities of AB-1 and cyflumetofen together with three other acaricides were tested against Drosophila.Of the compounds tested,the toxicity of cyflumetofen and bifenazate could not be measured in WT and mutant fly strains.Abamectin showed high toxicity but no selectivity against these WT and two mutant strains of flies.Interestingly,AB-1 and cyenopyrafen were not toxic to WT Drosophila,but did display higher toxicity to both the HLT-YT and HLT-LT strains,albeit still at high tested doses.This suggests that these two compounds or their metabolites can inhibit genome-modified Drosophila SDH and the mutations in this motif can conspicuously increase the sensitivity to SDHIs.The IC₅₀ values of AB-1 against HLT-YT（19.42 n M）and HLT-LT fly strains（62.26 n M）are～144 times and～45 times lower than the one against W1118strain（2.79μM）.In addition,the results from ATP quantification and relative respiration inhibition rate are consistent with the toxicity assays.All the above evidence supports the importance of the identified specific motif in the inhibitory activity of AB-1.We established the CRISPR/Cas9 gene editing technology system in T.cinnabarinus by knocking out the body color-related phytoene dehydrogenase Tc01g11270 gene successfully.However,it is a pity that no point mutation strain of T.cinnabarinus was obtained to verify the relationship between the difference of key amino acid motifs and the toxicity selectivity of cyflumetofen/AB-1.