Screening And Identification Of Pesticide-degrading Bacteria In The Gut Macro Composition And Anopheles Genome

Anopheles sinensis mainly breeding in paddy is the important malaria vector in vast plain area in China. The levels of mosquito insecticide resistance have been developed and promoted, due to the extensive usage of pesticides in paddies. The bacterial flora of degrading deltamethrin, trichlorfon and chlorpyrifos were screened and identified from paddy water, and degradation efficiency was detected in this study. The results hope to provide basic information for degrading pesticides residue in field paddies.The results were as below: firstly, bacterial flora of degrading deltamethrin, trichlorfon and chlorpyrifos were obtained using the classic method. Secondly, bacterial flora was identified by molecular marker of16S rRNA sequence. Genus Pseudomonas species could degrade both chlorpyrifos and trichlorfon, which suggested the degradation spectrum for organophosphorus was broadly. The three species were first reported of degradation chlorpyrifos and trichlorfon, as Klebsiella pneumonia, Rhodococcus ruber and Cupriavidus sp. Ten genera of deltamethrin-degraded flora were obtained, with genus Acinetobacter, Klebsiella, Citrobacter, Stenotrophomonas, Pseudomonas, Amycolatopsis, Rhodococcus, Ancylobacter, Ochrobactrum and Xanthobacter. The Acinetobacter and Klebsiella of them were dominant. There were six genera were first reported of deltamethrin-degraded bacterial flora, as Citrobacter, Xanthobacter, Amycolatopsis, Acinetobacter, Ochrobactrum and Ancylobacter. Finally, degradation efficiency of3pyrethroid insecticides was detected under laboratory conditions. The results showed that degradation rates continued to increase from2to8day, and the values were the highest on8day, with77%(bifenthrin),61%(deltamethrin) and74%(cyhalothrin), suggested that there was a broad-spectrum of this flora to degrade pyrethroid insecticides.As we known, the malaria parasites need to process a series of complicated development and reproduction in the Anopheles vector before infecting human. The bacteria in Anopheles mosquito gut could affect the malaria parasite development. In present study, the composition of metagenomic communities in field-collected An. sinensis and An. lesteri gut were investigated and analyzed by high-throughput sequencing technology, and the results will lay the foundation for clarifying the Anopheles-Plasmodium-bacteria interaction.The main results obtained were as follows:Ten samples including the breeding water, An. sinensis larvae and adults (blood-feeding and unblooded) and An. lesteri larvae were detected. Rarefaction curves and Alpha diversity indexes showed that the sequencing depth was adequate, and assessed OTU numbers should be close to the actual. The abundance of Phylum Proteobacteria was the highest in the mosquito gut, whether life stages or species. The3phyla of Firmicutes (39%), Bacteroidetes (34%) and Proteobacteria (25%) were dominant in paddy water. The comparison result of bacterial abundance differences among samples showed as below:At the phylum level, there was only one Phylum Proteobacteria different significantly between blood-feeding with unblooded adults, and9phyla different significantly between paddy water with blood-feeding adults, and with larvae. At family level, bacteria structure difference between adults and larvae was greater than blood-feeding with unblooded females, but the result of compared mosquito with water, the difference level among stages of mosquito was lower. Genera of sequences number above500were analyzed and showed that Acinetobacter, Thorsellia, Brucella, Pantoea and Pseudomonas occurred in5or more samples, among which Genus Acinetobacter species was present in all samples.