MIDGUT BACTERIAL DIVERSITY ANALYSIS OF LABORATORY REARED AND WILD Anopheles gambiae AND Culex quinquefasciatus MOSQUITO VECTORS IN AHERO, KENYA

Abstract

Mosquitoes transmit a wide range of pathogens that cause diseases in human and other animals. Midgut symbiotic bacteria are known to play fundamental roles in the biology of mosquitoes, however knowledge of midgut bacterial communities associated with mosquitoes is scanty due to limitation of the isolation techniques based on culturing. The available culture techniques reduce the chances in determination of the microbial diversity, since they sometimes miss out on non-culturable microbes. High throughput methods that involve direct isolation and analyses of nucleic acids from samples have been found more feasible in microbial diversity studies. The main objective of this study, was the application of metagenomics to study the composition and diversity of midgut bacteria in field collected and laboratory reared adult female Anopheles gambiae and Culex quinquefasciatus mosquitoes. Female adult mosquitoes were dissected and their total microbial deoxyribonucleic acid (DNA) isolated from the pooled midgut extracts by using Purelink genomic DNA mini kit (Invitrogen). The 16S rRNA gene variable regions V4 of the extracted DNA were amplified. Library construction was performed following Illumina sequencing protocol. Sequences were analyzed using QIIME pipeline, taxonomy was assigned using BLASTn against SILVA 119. The R programming language and Vegan package were used to calculate Bray-Curtis dissimilarities between datasets, hierarchical clustering and diversity indices. Phylogenetic analysis was done using MEGA 6.0 software. Results showed that 145 operational taxonomic units (OTUs) were realized at 3% genetic distance based on 16S rRNA gene sequence. The 145 OTUs spanned 12 phyla; Proteobacteria, Firmicutes, Bacteriodetes, Actinobacteria, Eukaryota, Gemmatimonadetes, Spirochaetae, Verrucomicrobia, Chloroflexi, Acidobacteria, Archeabacteria, Cyanobacteria and the no blast hits. Microbial community composition based on OTUs showed significant difference between field collected and laboratory reared mosquitoes (ᵪ2=45.0799, p=3.2 x 10-5). Similarly, there was a significant difference in community composition at OTU level between Anopheles gambiae and Culex quinquefasciatus (ᵪ2=31.2257,p=7.7 x 10-4). This study demonstrates a high microbial composition and diversity among field collected Anopheles gambiae and Culex quinquefasciatus than the laboratory reared mosquitoes. The bacterial composition and diversity appeared to be influenced by the environment and the species of the mosquitoes.