| Tetranychus cinnabarinus（Boisduval）is a worldwide agricultural pest which can feed on more than 1000 species of plants including cotton,corn,soybeans,fruits,and flowers,causing serious economic losses in agricultural production every year.Due to the biological characteristics of T.cinnabarinus such as short life cycle,strong reproductive ability and arrhenotokous parthenogenesis,as well as long-term irrational pesticides use,the problem of resistance is increasingly serious and field control is difficult.At present,chemical control is still the most effective control method for T.cinnabarinus.Etoxazole,as a diphenyl oxazoline derivative,is widely used to control the spider mite in the field.It is highly effective in egg killing,can control the entire juvenile stage of spider mite and make adult mite produce sterile offspring.Meanwhile,it also has certain activity on other arthropods,but relatively safe for human,livestock and other non-arthropods.Etoxazole is a chiral pesticide with two enantiomers,which has enantioselectivity in environmental behavior,bioactivity and toxicity.In this study,based on the bioactivity differences between different enantiomers of etoxazole towards T.cinnabarinus,we intend to investigate the mechanism from two aspects of drug metabolism and target binding.The main findings are as follows.1 Bioassay and phenotypic observation of T.cinnabarinus treated with etoxazole enantiomersBioassay results confirmed that the etoxazole enantiomers had significantly different toxicity to T.cinnabarinus,among which（+）-S-etoxazole was the effective enantiomer,（-）-R-etoxazole was the ineffective one.The bioactivity of（+）-S-etoxazole was 279.6times of（-）-R-etoxazole and 2.6 times of rac-etoxazole to the eggs of T.cinnabarinus,indicating that the toxicity is basically derived from the effective monomer.For adult mites,etoxazole had no significant effect on the amount of eggs laying,but the laid eggs could not hatch after treatment,and the hatching rate showed enantioselectivity.If the hatching rate of eggs laid on the same day reached the same level,the concentration treated by the ineffective enantiomer was about 5 times of that of the effective enantiomer.The phenotype showed that the treated eggs of T.cinnabarinus could develop to the red-eye stage and some even have the shape of juvenile mites,but eventually failed to hatch leading to death.2 The metabolism ability determination of T.cinnabarinus to etoxazole enantiomers and transcriptome sequencing analysisHigh performance liquid chromatography（HPLC）was used to determine the metabolizing ability to etoxazole enantiomers in the metabolizing enzyme solution of T.cinnabarinus eggs.The results showed that there was no significant difference in the etoxazole enantiomer content after metabolic reaction,and the metabolic rate was low（1.04%-8.40%）.The analysis of transcriptome data of T.cinnabarinus eggs treated with LC20 etoxazole showed that the response genes between the inactive and the control group,and between the active and the racemic group were close.The number of differencially expressed genes（DEGs）between these two groups were the least.15 P450s genes were annotated in DEGs between two enantiomers group（only 1 of which was upregulated）,but there were more than 20 genes related to chitin binding,such as chitinases（CHTs）and cuticular protein analogous to peritrophins 1-A（CPAPs-1）genes.Combined the transcriptome sequencing analysis with crude enzyme metabolism results,it was indicated that there was no significant difference in the metabolism of etoxazole enantiomers in the T.cinnabarinus eggs.However,more chitinin-related genes were differentially expressed,suggesting that there might be differences at the target bingding level.3 Cloning and sequence analysis of Chitin synthetase 1 gene（Tc CHS1）of T.cinnabarinusThe full length of Tc CHS1 coding region was 4614 bp,the number of encoded amino acids was 1537 aa,the relative molecular weight was 175.5 k Da,and the isoelectric point was 6.26.The amino acid sequences of CHS1 were analyzed,and the characteristic motifs EDR and QRRRW sequences were found.The TMHMM software was used to predict the structural domain of Tc CHS1,and it was found that Tc CHS1 had 18 transmembrane helix structures.The expression pattern of Tc CHS1 was conducted by RT-q PCR.The expression level of Tc CHS1 was high in all mite states,but it was highest in egg stage and lowest in adult mites.4 Molecular docking analysis and determination of chitin content in T.cinnabarinus treated with etoxazoleThe results of molecular docking showed that the binding affinity of Tc CHS1 with etoxazole enantiomers was different,and the binding affinity of the active monomer（+）-S-etoxazole（docking fraction:-7.404 kcal/mol）was stronger than that of the inactive body（-）-R-etoxazole（docking fraction:-6.850 kcal/mol）,suggesting that the reason for the difference in the toxicity of etoxazole enantiomers to T.cinnabarinus might be related to the binding difference to the target CHS1.The chitin content of T.cinnabarinus treated with etoxazole was determined,and it was found that under the same treatment concentration（25 mg/L,50 mg/L）,the chitin content of active enantiomer（+）-S-etoxazole treatment group was less than that of rac-etoxazole treatment group than that of ineffective monomer treatment group.Furthermore,the chitin content decreased with the increase of treated etoxazole concentration.These results indicated that the bioactivity difference of etoxazole enantiomers was closely related to the ability of enantiomers to inhibit CHS1.In this paper,it was confirmed that there was significant difference in the bioactivity between etoxazole enantiomers to T.cinnabarinus,and it was preliminarily proved from two aspects of detoxification metabolism and target binding,which the different binding ability of etoxazole enantiomers to the target Tc CHS1 was the main reason for the bioactivity difference.This study analyzed the toxicological mechanism of the bioactivity difference between etoxazole enantiomers against T.cinnabarinus at the molecular level,which provided a new research idea for the analysis of the mechanism of enantiomer bioactivity difference of chiral pesticides,and also laid a good research foundation for the molecular design of pesticides targeting chitin synthetase 1 and the large-scale application of the active etoxazole monomer. |
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