Insecticidal Mechanism Of Haedoxan A And The Resistance Mechanism Towards Aedes Aegypti

Phryma leptostachya was traditionally used as a natural insecticide to control houseflies and insect vegetables pests in East Asia.Haedoxan A(HA),which contains a3,7-dioxabicyclo[3.3.0]octane skeleton,has been proved to be the major insecticidal compound in P.leptostachya.Symptomatological observation indicated that HA-treated insect showed a series of nerve excitatory actions.Previous researches also demonstrated that HA could delay the decay rate of evoked excitatory junctional potentials from the Drosophila neuromuscular junction and sensory-central nervous system-motor circuit.Additionally,HA also induced a hyperpolarizing shift in the voltage dependence of inactivation of Drosophila melanogaster sensitive voltage-gated sodium channels(VGSCs).However,the insecticidal mechanism of HA still remains unclear so far.Hence,the insecticidal mechanism and target sites of HA were studied in this present research.On the one hand,themode of action of HA on VGSCs were explored and elucidated using two electrode voltage clamp technique and molecular biology methods,the target sites on VGSCs were discussed through source modeling and molecular docking tests;moreover,the mammalian cytotoxicity of HA was also investigated.one the other hand,an Aedes aegypti haedoxan A-resistant(HAR)strain was cultivated,which was used to systematically evaluate the toxicity and effect of HA on the population dynamics of Ae.Aegypti through bioassays and resistance risk assessment.Meanwhile,combined with proteomics technology,the mechanisms that underlying the resistance in HAR strain were discussed.Results were presented as follows:1.Electrophysiological experiments based on two electrode voltage clamp and Xenopus oocytes revealed that HA destroyed the normal nervous system function mainly by alter the kinetic characteristics of fast inactivation of insect VGSCs,causing the disorder of nervous system and resulting in neurotoxicity;according to site directed mutagenesis experiment,Ile at 265,Glu at 434 and Leu at 993 in VGSCs were binding sites of HA,or structurally coupling sites.Based on these results,the binding model of HA and Na_vPas-Based Bg Na_v 3D model was further optimized.The results showed that HA had multiple binding sites in the active cavity composed of IS4-S5 junction region and voltage sensing domain(VSD I and II)of VGSCs,which overlapped with two pyrethroid insecticides binding sites(?L45-?S5-?S6-?S6 and?L45-?S5-?S6-?S6).These results indicate that the binding sites of HA and pyrethroid insecticides on VGSCs are partially overlapped or allosteric coupled.2.HA did not alter the kinetic characteristics of rat sensitive VGSCs(r Nav1.2 and r Nav1.4)even at a larger dose which demonstrated that the VGSCs of mammals are not sensitive to HA;in addition,HA showed obvious selective toxicity to insects and mammals;The inhibitory effect of HA on proliferation of mammalian cells was also determined with permethrin as positive control.The IC₅₀ of permethrin to NIH-3T3 cells was 69.04?M,and to HEK293 cells was higher than 300?M.While the IC₅₀ of HA to HEK293 and NIH-3T3were both higher than 300?M,which indicated that HA had low cytotoxicity than permethrin and was relatively safe to mammals.3.Third instar larvae from the susceptible strain Waco were selected with HA at an interval under the selection pressure of 75%mortality,which has been propagated for 30generations.Compared with Waco,the resistant ratio of G₂₉ larvae from HAR strain was31.579-fold.The realized heritability of resistance to HA was relatively low,additionally,resistance risk assessment demonstrated that the LC₅₀ of HA to larvae increase by 10 times required 18 generations in the wild,which indicated that Ae.aegypti was not easily develop resistance to HA.4.The sublethal concentrations of PBO,DEM,and TPP were selected based on the LC₁₀ calculated from each strain,and results revealed that three inhibitors all showed obvious synergistic effect on HA.In the strain of HAR(G₁₉),the synergistic ratio of PBO,DEM and TPP towards HA was 16.580-,4.895-and 3.146-fold,respectively.Meanwhile,the synergistic effect of PBO on HA against the third instar larvae of Waco strain was8.675-fold.PBO is a special inhibitor of cytochrome P450 enzyme,combined with bioassay results,cytochrome P450 enzyme system not only participated in the metabolism and detoxification of HA in Aedes aegypti,but also undertook the HA resistance development in HAR strain.Cross-resistance tests used larvae from HAR showed that HA exhibited slightly cross-resistance to permethrin,cypermethrin and DDT,which resistance ratio was 4.763-,3.125-and 5.744-fold,respectively.Since HA,pyrethroids and DDT all targets at VGSCs,moreover,the act sites of HA and pyrethroids were overlapped,which may be contributed to the cross-resistance between HA and those two kinds insecticides.HA had low or no cross resistance with imidacloprid and chlorpyrifos,the resistance ratios were 1.901-and 1.023-fold,respectively.Moreover,HA was negatively crossed with carbamate insecticide indoxacarb and antibiotic insecticide avermectin,and the resistance ratio was–1.913-and–1.378-fold,respectively.5.Cytochrome P450 monoxygenases(CYP)was found overexpressed in HAR strain compared with susceptible Waco strain.Proteomic analysis revealed the expression of cytochrome P450 enzymes that contributed to the resistance development,such as p4504g15,p450 6a14 and p450 9f2,whcich belong to the CYP4,CYP6 and CYP9 family were up-regulated in the third-instar larvae of Ae.aegypti from HAR strain(G₁₉).Compared with Waco,these enzymes were up-regulated by more than 1.5 times at protein level,and 5-20 times at transcription level.Moreover,proteomics and real-time q PCR results demonstrated that the transcription levels of Cuticle Protein 19.8,Larval Cuticle A2B and Cuticle Protein 8 in the HAR strain(G₁₉)larvae were significantly up-regulated for 200-700 times than those of Waco.In addition,proteomic analysis and RT q PCR confirmed that the overexpression of Inhibitor of Growth Protein delayed the growth and development of the larvae from HAR strain which directly led to the prolongation of larval duration.In conclusion,the insecticidal mechanisms of HA on VGSCs were elucidated,the overlapped and allosteric coupling relationship between HA and pyrethroids on VGSCs was also discussed.HA was proved to have selective toxicity towards insect and mammalian VGSCs,and exhibited low cytotoxicity which indicated that HA was relatively safe to mammals.Enhanced metabolic enzymes activities and epidermal resistance was the mechanisms of resistance adopted by Aedes aegypti from HAR strain.Generally,these results provided useful information for the research and development of botanical insecticides with HA as the main insecticidal component or lead compound.