Bifenazate Resistance Selection And Resistance Mechnism Of Blattisocius Dentriticus

As one of Ascidae predator mite,Blattisocius dentriticus is commonly used to control stored grain pests,tea and citrus trees.Although,the predation range of this species is quite wide,chemical control is still indispensable in practice.To balance the conflict between chemical control and biological control,screening for the chemical-resistant predatory mite could be considered to be an option.Therefore,in this thesis,first,the potential control effect for Panonychus citri was estimated.After that,the bifenazate-resistant srtain of B.dentriticus was screened and obtained.The variation of cytb gene and the expressions of resistance related genes were analysized by SNP,high-throughput sequencing technology and qPCR.The results would provide a theoretical basis for the molecular transformation of predatory mites,especially the resistance mechanism.The main results were stated as follows: 1.Evaluation of the predation function of B.dentriticusThe predation function of B.dentriticus was determined with Aleuroglyphus ovatus.The result showed that the functional response was Holling type II.The predation ability of B.dentriticus increased with temperature rose within 16-28 °C and showed its distinctive ability in A.ovatus control,which indicated that alternative prey method could be used in large-scale breeding.2.The resistance selection of B.dentriticus against bifenazate and ecological fitness evaluationThe toxicity selection of B.dentriticus and P.citri was carried out,and the the toxicity selection index was 138.24,showing a high positive selectivity for the B.dentriticus and P.citri.After screening for several generations,the bioassay results showed that a low bifenazate-resistant strain of B.dentriticus was obtained,with the resistance ratio of 6.04-fold,compared to the susceptible strain.The lethal concentration 50(LC50)increased from 19,315.54 to 116,746.50 mg/L Besides,the cross-resistance of the bifenazate resistant strain of B.dentriticus was also studied.The results indcated that the bifenazate resistant strain of B.dentriticus showed cross-resistance with nitenpyram,amitraz and cyflumetofen,with the resistance ratios of 2.48,2.21 and 6.38,respectively.The predation functions to female mites and eggs of the P.citri were further studied.The results showed that there was no significant change in predation amount or functional response.These results indicated that resistance screening had no sigificant side effect on the predation function of the B.dentriticus.3.Cytb cloning and analysisA 1229 bp cDNA sequence was obtained,with its open reading frame 1083 bp,encoding 360 amino acids,naming as Bdbcytb.The homology comparison showed that the B.dentriticus cytochrome b gene was highly homologous to those in Galendromus Occidentalis and Phytoseiulus Persimilis.Random selected B.dentriticus(single mite)and specific primers were adopted in SNP analysis between the resistant strain and sensitive strain.However,no resistance-related mutations were found.In addition,DEF,DEM and PBO were applied in synergistic test.The results showed that the synergy ratios of DEM and PBO to the synergistic effect against bifenazate were 1.74 and 1.69 in bifenazate resistant and susceptible strains,respectively.The results indicated that glutathione S-transferase and multifunctional oxidase might involve in the metabolic detoxification of bifenazate.4.RNA sequencing and differentially expressed genes(DEGs)analysis of B.dentriticusIllumina sequencing technology and DEGs were applid to generate the transcriptom and differentially expressed genes between bifenazate resistant and susceptible strains of B.dentriticus.In total,108572 transcripts and 75697 Unigene were obtained,with 34912 annotated.For 3076 DEGs,174 were up-regulated and 2902 were down-regulated.Among the up-regulated genes,two genes related to resistance were identified,one was phosphoenolpyruvate carboxykinase-related gene(c20449.graph_c0)and the other was UDP-glucosyltransferase-related gene(c40332.graph_c1).5.Analysis of the differences in the expressions of metabolism-related genes and cytb gene of the B.dentriticusThe expression profiles the 10 detoxification enzyme-related genes and cytb genes were verified by real-time quantitative PCR.The results showed that CYP3A56 and GSTD1 were significantly up-regulated for 2.28 and 1.78 times in the bifenazate resistant strain(P < 0.05).Furthermore,the expression levels of these two genes and cytbgene were also estimated under the conditions at the concentrations of LC25,LC50 and LC75 for 12 h,24 h and 48 h.The data showed that the expressions of CYP3A56 were significantly increased under the conditions LC50 and LC75 for 12 h and 24 h.The expressions of GSTD1 were also up-regulated in different conditions(concentrations and time).Besides,the expressions of cytb were also up-regulated under LC25 and LC50 for 48 h.The results suggested that the three formentioned genes might involved in bifenazate resistant in B.dentriticus.