Effects Of Gut Microbiota Of Bactrocera Cucurbitae In The Metabolic Detoxification Of Emamectin Benzoate

Bactrocera cucurbitae(Coquillett)is an agricultural quarantine pest which mainly infests cucurbitaeceae crops.Chemical control is still the main control method at present,and the extensive use of insecticides inevitably led to the development of resistance,which makes the control of B.cucurbitae more difficult.There are many researches on the causes of insecticide resistance,but relatively few of them took gut microbiota into consideration related to the gut microbiota of insect.Therefore,in this thesis,two host populations of B.cucurbitae with different sensitivity to emamectin benzoate were selected as the research objects.Based on previous studies,the relationship between intestinal microorganisms of B.cucurbitae and resistance to emamectin benzoate was studied.The main research results are as follows:(1)The gut microbiota structure of adult B.cucurbitae of two host populations was compared before and after the treatment of emamectin benzoate.The gut microbiota structure of B.cucurbitae was analyzed by high-throughput sequencing.The results showed that the gut microbiota of C.sativus population and B.hispida population were mainly composed of Proteobacteria and Bacteroidetes at the phylum level.At the genus level,the dominant genus was Providencia,Enterobacter,Citrobacter,Klebsiella.It showed that no apparent changes of the dominant bacteria were observed in the pre-and post-treatments of emamectin benzoate.However,there were significant differences in the gut microbial community structure of B.cucurbitae adults between pre-and postemamectin benzoate treatments.(2)The traditional isolation culture method was used to isolate and purify the intestinal microorganisms of B.cucurbitae,and 16 S rDNA sequencing was performed for single colony.Combined with the two sequencing results,23 functional bacteria were screened,mainly belonging to Providencia,Citrobacter,Enterobacter,Klebsiella,Pectobacterium,and Raoultella.(3)The microbial culture method and q-PCR for 16 S rRNA gene were combined to evaluate the microbial clearance effect of mixed antibiotics on the gut microbiotal clearance of B.cucurbitae.It suggested that the intestinal bacteria of B.cucurbitae adults were almost eliminated after fed with 300 μg/mL mixed antibiotics for 5 days.(4)The results of emamectin benzoate bioassay showed that,the survival rates of B.cucurbitae inoculated with Providencia sp.(EC3 and EB3),Pectobacterium sp.(EC24),Raoultella sp.(EC94 and EC15),Citrobacter sp.(EB59 and EB26),and Klebsiella sp.(EC39)were significantly higher than the enteric sterility of B.cucurbitae group.The survival rates of B.cucurbitae were most significantly enhanced in the inoculation of EB3,EB5,EB26,EC94,and EC15 respectively.(5)Glutathione S-transferase activity and carboxylesterase enzyme activity of B.cucurbitae treated with emamectin benzoate were measured.The results showed that,the activity of GST and CarE enzyme in Citrobacter sp.EB59 and Citrobacter sp.EB26,Raoultella sp.EC94 and Raoultella sp.EC15,Providencia sp.EB3 were significantly higher than those in CON group.It indicated that these functional bacteria might increase the drug resistance of B.cucurbitae by increasing the activity of detoxification enzymes.In conclusion,the dominant genus in the midgut of B.cucurbitaee was Providencia,Enterobacter,Citrobacter,Klebsiella,and there were significant differences in the gut microbial community structure of B.cucurbitae adults between pre-and post-emamectin benzoate treatments;Gut microbiota may be related to the development of emamectin benzoate resistance in B.cucurbitae,and some gut microbiota may participate in the detoxification metabolism of emamectin benzoate in B.cucurbitae by increasing the activities of GST and CarE in vivo.