Study On The Mechanisms Of Phosphine Resistance In Tribolium Castaneum(Herbst)Based On CYP345 Family

Phosphine fumigation was used worldwide to control pest population in commodities such as stored grains and grain products.It was generally assumed that phosphine killed insects rapidly at a relatively low cost,leaving minimal residue on treated products.However,the intensive use of phosphine had led to stronger resistance in many insect pests especially the red flour beetle,Tribolium castaneum,one of the most serious stored-grain pests on agriculture crops.The red flour beetle enjoys worldwide distribution and developing strong resistance to phosphine in many countries,the resistant strains who survived the phosphine fumigation was considered to possess some specific capacity to detoxify phosphine,the degree to which individual insects could metabolize and detoxify this highly toxic chemical is of crucial importance for their survival in a chemically unfriendly environment,it is considered that the metabolic detoxification of insecticide was associated with the increased protein production and activities of these enzymes through the transcriptional up-regulation of the genes in insecticide resistant insects.But the mechanism of detoxification of phosphine in T.castaneum was still unclear.Cytochrome P450 is known to have a wide range of substrates and therefore has a greater role in the metabolism and detoxification of various xenobiotics.The most significant mechanisms involved in insecticide resistance are increased metabolic detoxification by cytochrome P450s,gene expression studies which focused on whole genome sequence analysis also suggested that the interaction via co-overexpression of multiple P450 genes may be responsible for the enhanced insecticide resistance.Recent transcriptome analysis about phosphine-resistant and-susceptible Tribolium castaneum has proved that phosphine could significantly induce P450 genes and 44 genes encoding CYP genes was increased in phosphine-resistant compared to-susceptible strain and five that were significantly increased under phosphine induction.Thus,the CYP345A1 which was one of these five significantly increased genes and the CYP345 family which contained six genes totally were selected and further identified.There were thirty five field T.castaneum populations which shared different phosphine resistance levels were collected from China.Finally,ten strains which showed a larger population density were selected for bioassays,specific activity assays,gene expression analysis and functional studies(RNA interference).The objective of this research was to identify and characterize the CYP345 family,evaluate its role in phosphine detoxification in Tribolium castaneum and provide better strategies for future pest management control.As a result,protein assays indicated that specific activity of Cytochrome P450 in strong resistant T.castaneum strains was significantly higher than susceptible strains,so did the medium resistant strains in numerical relativity,the result was similar to these previous research and indicated that Cytochrome P450 was an important detoxification enzyme in T.castaneum.Furthermore,result that specific activity of Cytochrome P450 significantly increased in two resistant strains but decreased in susceptible strain after phosphine induction suggested that Cytochrome P450 was reasonable for phosphine resistance in T.castaneum.Identification of the expression difference of CYP345 family between different ten strains indicated that only CYP345A1 and CYP345A2 were over-expressed in resistant strains;CYP345B1,CYP345C1 and CYP345D1 were not significantly different among different strains.Thus,we concluded that CYP345A1 and CYP345A2 contributed to phosphine resistance in T.castaneum,and phosphine induction was conducted in three T.castaneum strains to prove our hypothesis.Taken together,result showed that expression of 345A1 was obviously and rapidly up-regulated in strong resistant strain after phosphine induction at lh and 2h,and CYP345A2 was obviously up-regulated in susceptible strain at 6h and 12h after phosphine exposure,so did medium resistant strain,which implied that both CYP345A1 and CYP345A2 play important roles in P450-mediated resistance against phosphine in T.castaneum.Besides,the function of CYP345D2 should be further examined because the expression was higher in susceptible strain before phosphine induction.But there could be another possible reason which caused the up-regulation of these two genes.Because previous studies had reported that CYP genes of insect could be significantly up-regulated through insecticide induction in resistant strain,while the situation in susceptible strain was uncertain.For example,Li reported that fenpropathrin induction could significantly up-regulated NADPH-cytochrome P450 reductase in both resistant and susceptible Trtranychus cinnabarinus strains,while the expression of CYP389B1 and CYP392A26 showed no significant difference before and after fenpropathrin induction in susceptible strain.Zhu reported that CYP4G2,CYP4D4v2 and CYP6A38 could be induced only in the resistant D.melanogaster while showed no significant difference in other two susceptible strains.In this study,the susceptible individual was killed by phosphine while other resistant part survived,and survival part showed a higher expression of target genes as a result.However,more studies are needed to confirm it.Based on the result,we conclude that CYP345A1 and CYP345A2 could effectively contribute to phosphine resistance in T.castaneum and can be induced by phosphine in both resistant and susceptible strains,we further hypothesized that the resistant strain was derived from susceptible individual who survived after environmental stress especially insecticide fumigation such as phosphine exposure since the overexpression of resistance-related genes before and after insecticide exposure could possibly contribute to a high level of specific activity of detoxification enzymes and caused enhanced metabolic detoxification as a result.Previous work has revealed that RNA interference via dsRNA injection is more robust and systemic in T.castaneum,with silencing effective for longer periods and higher rates of phenotypic change.In current research,RNA interference was conducted to further investigate the function of CYP345A1 and CYP345A2.As expected,the expression of target gene was suppressed without significant off-target effects and the sensitivity to phosphine was increased after dsRNA treatment,and the mortality was increased significantly as a result.It was concluded that both CYP345A1 and CYP345A2 were core genes which played important roles in phosphine resistance.RNA interference could be used as a novel method of pest management and may be more effective than other pest control measures once the problems of efficiency and specificity in gene silencing are solved.In this study,there is an interesting result that although gene of CYP345A1 was significantly suppressed,the expression of CYP345A1 was resurging over time while CYP345A2 increased or decreased barely.Based on the result,we assumed that the suppression of CYP345A2 may be more effective in T.castaneum pest management for long periods but not interference CYP345A1.Further study should be performed to prove our hypothesis.Besides,although our data revealed the major role of CYP345A subfamily in phosphine resistance in T.castaneum,the contribution from other detoxification genes to phosphine could not be ignored,because the resistant strain was not killed completely after dsRNA-aided knockdown and the expression of CYP345A1 and CYP345A2 were not block absolutely,one or more of the other overexpressed genes in resistant strains may play minor roles in the development of phosphine resistance in T.castaneum.In conclusion,our results revealed that both the increased level of P450 activity and overexpression of resistant genes contribute to phosphine resistance in T.castaneum,and we identified six CYP345 family genes and implied that CYP345A1 and CYP345A2 were the core genes which caused phosphine resistance through quantitative RT-PCR and RNA interference.We further hypothesized that the mechanism of phosphine resistance was due to rapid stress reaction including gene and protein level.These findings may help researchers to formulate better strategies for effective pest management of Tribolium castaneum.