| Objective Anopheles mosquitoes are vectors of malaria,and a common vector control measure is the use of chemical insecticides.When Anopheles mosquitoes in chemical insecticide control programs encounter insecticide resistance constraints,new vector control programs are urgently needed.A paratransgenesis approach by engineering symbionts to express antimalarial effector has been suggested as a potential biological control strategy for malaria control after they have been reintroduced into the body using genetic engineering.At present,there are not many reports about the midgut symbionts of the native Anopheline species in China,so studies on the diversity of the midgut symbionts of Anopheles will be carried out,which will help to further understand the interactions between Anopheles and all three of their symbionts and pathogens,and provide a theoretical basis for the development of paratransgenic organisms to prevent malaria transmission.Methods I.Field mosquito trapping: captured Anopheles were morphologically taxonomically characterized by developing mouth aspirator and ultraviolet light traps method on animal rings and human rooms at 11 sampling sites in three border county areas,including Yingjiang County,Mengla County,and Jinping County,Yunnan,China.II.Symbiotic bacteria isolation and identification: the medium gut cultivable symbiotic bacteria of female adult Anopheles mosquitoes in the border area of Yunnan Province were isolated and cultured using L-B plates,and the main taxa of the medium gut cultivable symbiotic bacteria of Anopheles mosquitoes were identified by 16S rDNA sequencing.III.Diversity analysis of midgut symbionts: 16S rRNA amplicon analysis was applied to study the compositional diversity of midgut symbionts in female adult Anopheles mosquitoes at the border of Yunnan Province.Results A total of 505 female adult mosquitoes were collected at 11 sampling locations in the three border county regions of Yunnan Province belonging to fourteen species: Anopheles sinensis,Anopheles minimus,Anopheles vagus,Anopheles peditaeniatus,Anopheles tessellatus,Anopheles kunmingensis,Anopheles kochi,Anopheles pseudowillmori,Anopheles argyropus,Anopheles crawfardi,Anopheles barbirostris,Anopheles splendidus,Anopheles culicifacies,and Anopheles jeyporiensis.A total of 381 bacteria were isolated and purified from the midguts of nine female adult Anopheles mosquitoes,and after selection,species information was obtained for 234 bacteria.234 bacterial strains belonged to four phyla,Proteobacteria,Actinobacteria,Firmicutes and Bacteroidetes,respectively,of which Acinetobacter and Pseudomonas were the most dominant species,accounting for 34.2%(80/234)of the total number of identified bacteria.The midgut DNA of eight Anopheles from three regions was sequenced and analyzed for 16S rRNA amplicons,yielding a total of 360487 sequences for analysis and 5675 ASVs for species annotation after denoising.The average number of ASV in the Anopheles kochi in Mengla County was as high as 260,whereas the average number in the Anopheles sinensis in Mengla County was the lowest at only 41.The Rarefaction Curves indicated that the sequencing depth was adequate and the number of ASVs was sufficient to reflect the diversity level of bacteria in this study,and the Rarefaction Curves in Jinping County and Mengla County(except for Anopheles sinensis and Anopheles vagus)were both above that in Yinjiang County,where the symbiotic bacteria abundance in the Anopheles midgut was generally higher than that in Yinjiang County.At the phylum level,a total of 25 phyla of bacteria were detected,and Proteobacteria,Firmicutes,Bacteroidetes,and actinobacteria were also the dominant taxon.At the genus level,the species of gut symbionts in Anopheles in the field were quite diverse,and the relative abundance of bacterial symbiont in all species of Anopheles in the three regions was high(Escherichia-Shigella,Lactobacillus,Pseudomonas).The results of the analysis of the significant difference in the diversity of bacterial symbionts in the midgut were as follows:(a)The composition of the midgut symbiont of Anopheles sinensis differed significantly among the three regions.The genera Arcobacter,Lactobacillus,Escherichia-Shigella and Pseudomonas played an important role in the significant difference in the abundance of symbiotic bacteria between Anopheles sinensis in Mengla County and Yingjiang County.The diversity of midgut commensals in Anopheles sinensis was similar between Yinjiang County and Jinping County,but the relative abundance of commensals differed.(b)The bacterial symbionts diversity in the midgut of Anopheles sinensis in Mengla County was significantly lower than that of Anopheles sinensis in Yinjiang and Jinping Counties.(c)Anopheles midgut symbiotic bacteria composition is similar among different Anopheles species in the same region.Conclusions Through isolation,cultivation,and analysis of 16S rDNA sequences,234 cultivable bacteria were successfully identified in nine Anopheles midguts,and a midgut cultivable bacterial symbiont resource database derived from field population of native Anopheles in Yunnan Province was established.Midgut symbiotic bacteria diversity information was obtained for 8 Anopheles by 16S rRNA amplicon.It provides a foundation for further studies on the function of gut symbionts in Anopheles and for the development of paratransgenic controls of malaria. |
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