The Mechanism Of Selective Toxicity Of Cyflumetofen Between Neoseiulus Barkeri And Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite,Tetranychus cinnabarinus?Boisduval?,is one of the most important agricultural pests worldwide,which can feed on cowpeas,eggplants,cottons,flowers and other economical plants.Because of its high fecundity and short lifecycle,T.cinnabarinus has developed serious resistance to most acaricides under persistent abuse of chemical toxicants.Neoseiulus barkeri?Huges?is an excellent biocontrol agent and widely distributed because of its rapid development rate and high fecundity.It is commercially produced and applied in greenhouse and orchards.Nowadays,it is considered as one of the best natural enemies for biological control.Cyflumetofen has shown excellent efficacy for control of spider mites and it was reported that cyflumetofen was safe to non-targeted organisms.In this study,the selective toxicity of cyflumetofen on the two species of mites was studied based on extremely different performance of cyflumetofen on T.cinnabarinus and N.barkeri.The main contents and results are as follows:1.Study on the toxicity difference of acaricides to T.cinnabarinus and N.barkeriBioassy tests were carried out against the female adults of T.cinnabarinus and N.barkeri by the method of modified residue coated vials?RCV?.The results showed that the LC₅₀0 of cyflumetofen to T.cinnabarinus was 7.816 mg/L,but the LC₅₀0 of cyflumetofen to N.barkeri was more than 100,000 mg/L,the insensitivity of predator mite was 12,794 times higher than that of pest mite;the LC₅₀0 of cyenopyrafen to T.cinnabarinus was 4.587 mg/L,but the LC₅₀0 of cyenopyrafen to N.barkeri was more than 50,000 mg/L,the insensitivity of predator mite was 10,900 times higher than that of pest mite;the LC₅₀s of fenpropathrin to T.cinnabarinus and N.barkeri were 606.98mg/L and 140.923 mg/L,respectively,and the insensitivity of predator mite to fenpropathrin was 0.23-fold than that of pest mites;the LC₅₀ of malathion to T.cinnabarinus and N.barkeri were 4112.088 mg/L and 65.594 mg/L,respectively,showing that N.barkeri was 0.02 times more insensitive than T.cinnabarinus.Synergism experiments showed that three synergists?PBO,DEM and TPP?had different effects on T.cinnabarinus and N.barkeri.The synergism rations were 1.43?1.86 and 2.33 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.The bioassay results showed that there were a significant toxicity differences between T.cinnabarinus and N.barkeri.The bioassay results of pesticides and synergists indirectly showed that the epidermal and metabolic enzymes were not the main reasons for the toxicity difference of cyflumetofen to T.cinnabarinus and N.barkeri.And it was strongly suggested that target difference was the main reason for the excellent selectivity of SQR inhibitors between the two species.2.Detoxifying enzyme activity of T.cinnabarinus and N.barkeriOn the basis of synergism experiments,the activity differences of three detoxifying enzymes of T.cinnabarinus and N.barkeri were further detected and compared.The results showed that the activities of GSTs and P450s of T.cinnabarinus were significantly higher than those of N.barkeri,and the activity of CarEs was significantly lower than that of N.barkeri.The changes of enzyme activity of T.cinnabarinus induced by different concentrations of cyflumetofen(LC₁₀,LC₃₀ and LC₅₀)were as follows:CarEs activity increased significantly after induction by LC₃₀,GSTs activity decreased significantly after induction by LC₅₀ and LC₃₀,while the activity of other induction treatments did not change significantly.For the N.barkeri,the activity of P450s increased significantly after being induced by LC₅₀ and LC₃₀,but there were no significant changes in other induction treatments.The results of detoxification enzyme activity tests showed that three detoxification enzymes and their response to the stress of N.barkeri were not stronger than those of T.cinnabarinus.In turn,the reason why the insensitivity of N.barkeri to cyflumetofen was more than 12,794 times higher than that of T.cinnabarinus was not related to the difference of detoxification metabolic enzyme activity.3.Activity of Mitochondrial Complex II and ATP content in T.cinnabarinus and N.barkeriThe activity of mitochondrial electron transfers chain complex II?SQR,target of cyflumetofen?of T.cinnabarinus and N.barkeri showed that the SQR activity of N.barkeri was 1.63 times higher than that of T.cinnabarinus.The half maximal inhibitory concentration(IC₅₀)of cyflumetofen on SQR activity in vitro of T.cinnabarinus and N.barkeri were 23.141 nmol/L and 4,531,160 nmol/L,respectively.The insensitivity of cyflumetofen to SQR to N.barkeri(N.b IC₅₀/T.c IC₅₀)was195,807 times higher than that of T.cinnabarinus.It strongly suggested that the toxicity difference of cyflumetofen to T.cinnabarinus and N.barkeri was related to the target difference.The main biological function of mitochondrial complex II?SQR?is to participate in the generation of ATP.The ATP content of T.cinnabarinus and N.barkeri was detected and compared by ATP detection kit.The results showed that the ATP content of T.cinnabarinus was 4.376 nmol/mg Pro,and that of N.barkeri was 13.594 nmol/mg Pro.T.cinnabarinus and N.barkeri were treated with 10 mg/L cyflumetofen for 4hours respectively.After the treatment,the ATP content of T.cinnabarinus decreased significantly,but there was no significant change of the ATP content in N.barkeri,which further indicated that the different binding inhibition by cyflumetofen between the two mites was due to the SQR structure difference.4.Cloning of Mitochondrial Complex II Genes and active sites analysis of T.cinnabarinus and N.barkeriThe SQR gene fragments of T.cinnabarinus and N.barkeri were obtained by analyzing the transcriptome data.According to the obtained gene fragments,five SQR genes of T.cinnabarinus and N.barkeri were cloned by RACE PCR,which were named as NbSDHA?NbSDHB?NbSDHC?NbSDHD?NbSDH5 and TcSDHA?TcSDHB?TcSDHC?TcSDHD?TcSDH5 respectively.Functional regions of each subunit were obtained by NCBI BLAST.Amino acid sequence alignment of functional regions of each subunit showed that there were as many as 220 different sites.These differences indicated that there were significant differences in SQR between T.cinnabarinus and N.barkeri.These differences might be the main reason for the difference in toxicity caused by the difference in the binding ability between T.cinnabarinus and N.barkeri.On the one hand,these differences indicated that there were great differences in the structure of SQR between the two species,on the other hand,these differences might be the main reason for the huge toxicity difference between the two mite species.