Study On Synergism Of Phoxim With Cry1Ca And Its Synergistic Mechanism In Spodoptera Exigua

Spodoptera exigua is an intermittent outbreak of polyphagous pests and the damage in cotton fields has become more and more serious since the late 1980 s.However,the prevention of S.exigua relies mainly on the chemical prevention,causes resistence to pesticides easily.Phoxim is an organophosphate insecticide,which is high efficiency and broad spectrum,and is widely used to prevent chewing mouthpart pests.Cry1Ca is a kind of microbiological insecticidal protein with high activity against S.exigua.Combination of pesticides has the advantages of broadening the spectrum of prevention,synergies,and delaying insecticide resistance.Therefore,the purpose of this research was to study the virulence changes of phoxim and Cry1Ca against S.exigua,clarify the synergistic effect of phoxim(LC₃₀)and Cry1Ca(LC₃₀)on toxicity of S.exigua,and explore the changes of detoxifying enzymes,resistance-related genes and symbiotic microorganisms caused by phoxim and Cry1Ca in S.exigua.The results of this study will provide theoretical basis for elucidating the synergistic mechanism of phoxim and Cry1Ca and the molecular mechanism of S.exigua resistance.The main results are as follows:1.Determination of virulence of phoxim and Cry1Ca against S.exigua by surface contamination.The 2^nd instar larvae of S.exigua fed on mixture of phoxim and Cry1Ca for 3 d,5 d and 7 d,the mortality were 52.73%,93.13% and 95.87%,respectively.The mortality of S.exigua fed with the mixture was 22.06%,48.46%,50.87% higher than that of the phoxim,and 52.73%,75.40%,78.14% higher than that of the Cry1Ca.Therefore,the virulence of the mixture is significantly higher than that of the single agents.Moreover,the larval duration of S.exigua feeding on the mixture was extended to 15.6 d,which prolonged 2.1 d and 4.7 d longer than that of the phoxim and Cry1Ca.Thus,phoxim can be mixed with Cry1Ca to improve the control effect against S.exigua..2.The mixtures affect the expression of alkaline phosphatase?ALP?of S.exigua.The full length sequences of Ses ALP1 and Ses ALP2 were obtained by the method of RACE,and the expression of Ses ALP1 and Ses ALP2 were aberrantly high in the larval midgut tissue.The effect of the mixture on the expression of Se ALPs m RNA were investigated by q PCR.The results showed that the expression levels of Ses ALP1 and Ses ALP2 were up-regulated and down-regulated after 32 h treatment,respectively.Within 0 h³2 h,the expression of Sem ALP1 increased at first and decreased subsequently,the expression level of Sem ALP2^Sem ALP5 decreased at first and increased subsequently.3.The mixtures have a great influence on the detoxifying enzymes and resistance-related genes of S.exigua.The larvae were feeding on 3 agents(phoxim(LC₃₀),Cry1Ca(LC₃₀)and phoxim + Cry1Ca)for 8 h,the CAT activity of S.exigua increased significantly,which was 37.74-fold,1.23-fold and 43.65-fold higher than control,and was significantly higher than that of the single agent.After treated 24 h with agents,The ACh E activities were significantly lower than control,but there are no significant in different treatments of agent.After feeding on Cry1Ca for 8 h and 24 h,the CarE activities were 4.97 and 1.42 times of control,however,and the inhibition rates of Car E activity of phoxim or the mixture were 37.72%,65.35% and 45.27%,52.36%,respectively.Therefore,the activity of Car E is induced by Cry1Ca,the activity of Car E was inhbited by phoxim and mixture.Using the q PCR,we analysed the changes of expression levels of P450 and ABCC from 0 h to 24 h,the results showed that the expression levels of ABCC3,ABCC4,and ABCC12 decreased first and then increased.Moreover,the expression levels of CYP4L7,CYP4M15,CYP4S9,CYP6AB31,CYP6AE47,CYP6AE97,CYP9A9,CYP9A10,ABCC1,ABCC2,ABCC5 and ABCC6 increased first and then decreased,subsequently.4.The mixtures have a great effect on the diversity of symbiotic microorganisms of S.exigua.Using 16 S r DNA metagenomic sequencing,we detected the synergistic effect of phoxim and Cry1Ca on the symbiotic diversity of S.exigua in vivo.The number of OTUs used for species classification was 4318.The dilution curves of RS1?phoxim + Cry1Ca?,RS2?phoxim?,RS3?Cry1Ca?,and RS4?control?samples gradually tended to be gentle,which basically reflect on the community composition of microorganisms of treated larvae.The results of OTUs showed that the abundance of bacterial community in four groups of samples was RS2> RS3> RS1> RS4.Moreover,we analyzed the differences between the beta diversity,the results indiced that the order of diversity of bacterial community in four samples was RS2> RS3> RS4> RS1.Therefore,the combination of phoxim and Cry1Ca significantly decreased the microbial diversity in S.exigua.There are 4 kinds of absolute dominant flora at the level of Phylum by differenr treatments,they are Firmicutes,Proteobacteria,Actinobacteria,Bacteroidetes.There are 6 types of absolute dominant bacteria at the level of Class by differenr treatment,they were Clostridia,unidentified-Actinobacteria,Gammaproteobacteria,Bacilli,Bacteroidia,Alphaproteobacteria.The results of the Genus level analysis indicated that the combination of phoxim and Cry1Ca significantly increased the abundance of Rhodococcus and significantly reduced the abundance of Lactobacillus and Acinetobacter.In summary,phoxim and Cry1Ca have a synergistic effect on the virulence of S.exigua,and the mixture has an influence on the activities of protective enzyme and detoxifying enzyme,the expression levels of resistance genes and the community structure of microbes in S.exigua.All of the above reasons could lead to the susceptibility change of S.exigua to the mixture.This research preliminarily investigated the virulence effects and synergistic mechanisms of phoxim and Cry1Ca on S.exigua,and the results of this study provides theoretical guidance for resistance management of S.exigua.