The Mechanism Of Difference In Acaricidal Activity Between Amitraz And Its Metabolite DPMF

Tetranychus cinnabarinus（Boisduval）has the characteristics of small size,fast reproduction speed,short life cycle and strong environmental adaptability.It can break out in a short time and cause serious economic losses.Amitraz is a formamidine acaricide,which is mainly used to control ticks,whiteflies,scale insects,etc.,and has good control effect on the mite stage of the whole development stage of spider mites.DPMF（N²-（2,4-Dimethylphenyl）-N¹-methyformamidine）is an active metabolite of amitraz and has better insecticidal activity against ticks.Amitraz is widely used to control phytophagous spider mites,while the acaricidal activity of metabolite DPMF has not been reported.Therefore,this study takes an important agricultural pest mite T.cinnabarinus as the research object.On the basis of the difference in acaricidal activity between amitraz and DPMF determined by indoor bioassay,the mechanism of difference was studied from two aspects of metabolism and target by synergist experiment,target sensitivity detection and molecular docking.The main results are as follows:1.Determination of acaricidal activity and synergist test of amitraz and DPMFThe toxicity of amitraz and DPMF to T.cinnabarinus was determined by the residual film method.The results showed that the medium lethal concentration(LC₅₀)of DPMF and amitraz against the T.cinnabarinus was 6.37 mg/L and 26.18 mg/L,respectively.The acaricidal activity of DPMF was better than that of amitraz,and the acaricidal activity of the former was about 4.11 times that of the latter.Three syner gists,piperonyl butoxide（PBO）,deltamethrin（DEF）and diethyl maleate（DEM）,were used to inhibit three detoxification enzymes（P450,CCE and GST）,respectively.The toxicity of the three synergists to T.cinnabarinus was determined by amitraz and DPMF.The results showed that the synergistic ratios（SR）of the three synergists PBO,DEF and DEM to amitraz were 0.96,0.97 and 1.08.The synergistic ratios（SR）of PBO,DEF and DEM to DPMF were 0.90,0.96 and 0.86,respectively.After inhibiting the three detoxification enzymes,the acaricidal activity of amitraz and DPMF did not change significantly,indicating that the difference in activity between the two may not be related to the difference in detoxification ability of metabolic enzymes.2.Sensitivity analysis of the Tetranychus cinnabarinus TcOctβ2R to amitraz and DPMFBased on the previous study that TcOctβ2R is the target of amitraz,the activity of agonist amitraz and DPMF in vitro was further determined by mammalian heterologous expression system and cyclic adenosine monophosphate（c AMP）assay.The results showed that both amitraz and DPMF could activate TcOctβ2R and induce intracellular c AMP production.The medium effective concentration(EC₅₀)of amitraz and DPMF were 75.3 n M and 8.71 n M,respectively.The agonistic effect of DPMF was about 8.65times that of amitraz.At the same time,the effects of octopamine（OA）and dopamine（DA）on the activity of TcOctβ2R were determined.The results showed that OA could also effectively activate TcOctβ2R and increase the intracellular c AMP level,but dopamine did not.The EC₅₀ of octopamine was 158 n M.According to the activation effect,DPMF>amitraz>octopamine,indicating that the metabolite DPMF can activate TcOctβ2R more effectively than the parent compound amitraz and the endogenous agonist octopamine.3.Analysis of the binding mode of TcOctβ2R protein of Tetranychus cinnabarinus with amitraz and DPMFThe three-dimensional structure of the T.cinnabarinus TcOctβ2R protein was modelled by SWISS-MODEL software based on the available sequences.Then molecular docking analysis of the TcOctβ2R protein model and the two ligand compounds were performed separately using Autodock.The results showed that the free binding energy of amitraz to TcOctβ2R was-5.78 Kcal/mol and that of DPMF was-6.23 Kcal/mol,indicating that DPMF had a stronger binding capacity to TcOctβ2R than amitraz,which was consistent with the bioassay results.Analysis of the binding modes showed that both amitraz and DPMF bound to TcOctβ2R by hydrogen bonding and hydrophobic interactions.Amitraz bound to Asp109,Ser113 and Try407 on TcOctβ2R by hydrogen bonding force;DPMF bound to Asn106,Ser187 and Lys189,respectively.It is speculated that the difference in binding sites is the reason for the different acaricidal activity of amitraz and DPMF.4.4.Functional analysis of octopamine receptor TcOctβ2R mutation in Tetranychus cinnabarinusThe TcOctβ2R gene of T.cinnabarinus was mutated by overlap extension PCR.The results of agarose gel electrophoresis and sequencing showed that the I39F mutation（homologous mutation of I61F in Rhipicephalus microplus）was successfully constructed in vitro.The mutant recombinant expression vector pc DNA3.1（+）-TcOctβ2R^I39F was constructed and transiently expressed in HEK-293 cells.The results showed that the sensitivity of HEK-293 cells transfected with mutant plasmid vectors to DPMF,amitraz and octopamine was reduced compared with that before mutation.The agonistic effects of DPMF(EC₅₀=16.9 n M),amitraz(EC₅₀=193 n M)and octopamine(EC₅₀=1208 n M)were reduced by about 1.94 times,2.56 times and 7.65 times,respectively.In addition,the sensitivity of DPMF before mutation was about 8.65 times that of amitraz,and the sensitivity of the two increased to 11.42 times after mutation.The free binding energy and interaction force of ligand and receptor binding were analyzed by molecular docking technology.The results showed that the binding ability of TcOctβ2R^I39F to amitraz（-4.04Kcal/mol）and DPMF（-4.68 Kcal/mol）decreased.TcOctβ2R^I39F and DPMF interact through hydrophobic force and hydrogen bond,and no hydrogen bond binding site with amitraz has been found.Therefore,it is speculated that I39F is not a selective binding site.In summary,this study determined the toxicological differences between amitraz and metabolite DPMF by indoor bioassay,followed by the analysis of the reasons for the differences at two levels,metabolism and target,and demonstrated that the difference in target binding capacity and binding sites were the reason for the differences,while detoxification metabolism was not.The results of the study will promote the understanding of the acaricidal activity of DPMF and the mutational function of the octopamine receptor of the T.cinnabarinus,provide a theoretical basis for the development of lead compounds acting on the octopamine receptor,and have important scientific significance for the design of new acaricides with high specificity.