Study On The Mechanism Of Action Of Haedoxan A

Haedoxan A is major component with higher insecticidal activity in Phryma leptostachya.Previous research results found that haedoxan A delayed the decay rate of excitatory junction potential?EJP?on Drosophila neuromuscular junction,increased the frequency of miniature excitatory junction potentials?mEJP?on Drosophila neuromuscular junction and changed the voltage dependence of inactivation of insect sodium channels,suggesting that haedoxan A may act on insect sodium channels.Some studies have shown that the presence of divalent cation channels in insects such as the DSC1 channel in the fly which may have a synergistic regulatory effect on voltage-gated sodium channels.Therefore,based on the four Drosophila strains-w¹¹¹⁸?wild type??DSC1^-/-?DSC1 knockout mututant??para^ts1?pyrethroid-resistant mutant?and para^ts1;DSC1^-/-?double mutant?,this paper conducted insecticide bioassays to measure the toxicity of haedoxan A to adults and larvae of tested insects and compared the effects of haedoxan A to the potentials of neuromuscular junctions of four Drosophila strains larvae by using intracellular microelectrode recorded technique.The main results are as follows:The toxicity experiment of four larval and adult Drosophila strains to haedoxan A showed that compared with w¹¹¹⁸,the resistance of adults and larvae of para^ts1 line to haedoxan A was-1.2 and-1.8 fold,separately.The resistance of adults and larvae of para^ts1line to permethrin was 2.1 and 11.2 times,individually,compared to w¹¹¹⁸.Therefore,it was shown that haedoxan A and permethrin had no cross-resistance and may have cetain negative cross-resistance.The susceptibility order of the four Drosophila adults to haedoxan A was para^ts1;DSC1^-/->para^ts1>w¹¹¹⁸>DSC1^-/-;The sensitivity of the four Drosophila strains larvae to haedoxan A was para^ts1>para^ts1;DSC1^-/->w¹¹¹⁸>DSC1^-/-.Among them,compared with w¹¹¹⁸,after knockout the DSC1 channel,adults and larvae of DSC1^-/-were less sensitive to haedoxan A.Furthermore,compared with para^ts1 larvae,para^ts1 strain larvae that were knocked out DSC1 channel were insensitive to haedoxan A,while adults of para^ts1 strain knocked out the DSC1 channel was more sensitive to haedoxan A,compared with para^ts1adults,indicating that when haedoxan A affected insect sodium channels,DSC1 channels played different regulatory roles on adults and larvae.After direct treatment of four Drosophila strains larvae with haedoxan A for 2 h,the changes in neuromuscular junction potentials were recorded by using intracellular microelectrode technique.The results showed that the amplitude and frequency of the spontaneous excitatory junction potential?mEJP?and the amplitude of the excitatory junction potential?EJP?of the four strains larvae were all obviously changed,especially the order of EJP amplitude of the four Drosophila strains which was consistent with the sensitivity order of the the four Drosophila strains larvae to haedoxan A.Further treatment of the neuromuscular specimens of four Drosophila strains with haedoxan A for 60 min,we found that haedoxan A could increase the amplitude of excitatory junctional potentials?EJPs?,delay the decay of excitability and increase the frequency of miniature excitatory junctional potentials?mEJPs?,except for the para^ts1;DSC1^-/-double mutant.The above findings further confirmed that haedoxan A affected neuromuscular synaptic transmission and enhanced synaptic excitability.In addition,based on that haedoxan A may act on the insect sodium channel and combined with the results of this paper,it was speculated that haedoxan A may have a unique binding site on the sodium channel that was allosterically coupled to pyrethroid binding site.Moreover the DSC1 channel may coordinate the regulation of insect sodium channels,resulting in maintaining synaptic stability and excitability in insects.Furthermore haedoxan A may also act on the DSC1 channel.As to how HA acts on sodium channels and the DSC1 channel,further studies are needed.