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.
