Study On Action Mechanism Of Emamectin Benzoate On Bursaphelenchus Xylophilus

Emamectin benzoate（EB）is a kind of 16-membered macrolide lactones,produced by fermentation of Streptomyces avermitilis with a 16-membered ring as its main structure.Its insecticidal activity is much higher than that of avermectin products,and its toxicity to mammals is low.Nowadays,it is one of the most widely used broad-spectrum insecticides with high efficiency and low toxicity.It is also one of the main compoundsused to control Bursaphelenchus xylophilus by trunk injection.However,its action mechanism on B.xylophilus has not been verified yet.In this study,genes encoding glutamate-gated chloride channel（GluCl）andγ-aminobutyric acid-gated chloride channel（GABACl）of B.xylophilus were screened and cloned,and the binding process between the two target sites and16-membered macrolide lactones was verified by RNAi.The major results are summarized as follows:1.The toxicity effect of EB against B.xylophilus was verified by a series of bioassays.The results showed that EB with 0.1 mg/L treatment can significantly reduce eggs laid per female nematode（F=145.25,P＜0.001）and thrashing frequency（F=141.97,P＜0.001）,inhibit the growing progress（F=175.75,P＜0.001）and had no significant effect on sex ratio（F=1.412,P=0.281）.These bioassay results elucidate the toxicity effect of EB against B.xylophilus from the perspective of reproduction,development and motor activity.2.Differentially expressed genes（DEGs）were screened by RNA-seq.The results showed there were only 7 DEGs between RNA-seq libraries of B.xylophilus treated by 0.1 mg/L EB for 12 h,and5741 DEGs were found after 24 h.Between EB24 and ON24,758 genes were up-regulated and 562genes were down-regulated.They could be divided into genes related to life cycle,nervous and motor system,xenobiotics biodegradation and pathogenesis.The quality of RNA-seq was verified by qPCR.Relative expression levels of DEGs encoding pectate lyase（F=151.9,P＜0.001）andβ-1,4-endoglucanase（F=735.2,P＜0.001）were down-regulated after EB treatments,and DEGs encoding glutamate-gated chloride channel（F=35.0,P＜0.001）,γ-aminobutyric acidβreceptor（F=15.8,P＜0.001）,UDP-glucuronosyl transferase（F=17.0,P＜0.001）and ATP-binding cassette transporter（F=23.1,P＜0.001）were down-regulated after EB treatments.They conformed to the RNA-seq data,which proved the correctness of RNA-seq.Bioinformatics analysis verify function integrity of screened chloride ion channels.BxGluCl and BxGABACl owned structure characteristics conformed to cys-loop LGICs.Phylogenetic tree constructions showed BxGluCl and BxGABACl had high homology with chloride channels from other nematodes.It inferred BxGluCl and BxGABACl owned functions similar to other chloride channels and could be expressed completely.3.Through PCR and recombinant plasmid construction,the target genes were cloned,and the expression levels of them at different B.xylophilus developmental stages and expression patterns after EB treatments were investigated by qPCR.The electrophoresis results of PCR products were in accord with expectations.A pGEM-T Easy plasmid was used to construct recombinant plasmid and introduced into Escherichia coli.The products were sent for sequencing,as the results were in accord with expectations.The fragment lengths were 1386 bp and 1446 bp respectively.The expression levels were relatively low at egg stage,which were high at J2-J4 stages and reached the highest at adult stage.After treatments of EB,the expression levels of BxGluCl（F=2.308,P=0.203）and BxGABACl（F=4.654,P=0.097）did not change significantly within 12 h,but increased significantly after 24 h（F=8.248,P=0.019;F=8.436,P=0.018）;The expression levels did not change significantly within 48 h after treatment with thiacloprid.4.The target sites of EB against B.xylophilus were verified by RNAi.The silencing efficiency of BxGluCl and BxGABACl in B.xylophilus adults reached 54.93%and 38.53%after 24 h dsRNA treatment,which were relatively high.Using EB,tenvermectin A,tenvermectin B,abamectin B_1a and thiacloprid in LC₅₀ and LC₈₀ concentrations,the results showed the sensitivity of B.xylophilus to16-membered macrolide lactones decreased significantly after RNAi,and the sensitivity of B.xylophilus to thiacloprid did not change significantly after RNAi;Meanwhile,the thrashing frequency of B.xylophilus also significantly reduced after silencing of the mixed dsRNA（dsMix）compoundsof BxGluCl and BxGABACl（F=6.310,P=0.036）.The results confirmed the binding sites of EB against B.xylophilus were BxGluCl and BxGABACl proteins.