Generational conservation of composition and diversity of field-acquired midgut microbiota in Anopheles gambiae (sensu lato) during colonization in the laboratory

Abstract

Background: The gut microbiota is known to play a role in a mosquito vector's life history, a subject of increasing research. Laboratory experiments are essential for such studies and require laboratory colonies. In this study, the conservation of field-obtained midgut microbiota was evaluated in laboratory-reared Anopheles gambiae (s.l.) mosquitoes continuously hatched in water from field breeding habitats.

Methods: Pupae and late instars were obtained from the field and reared, and the emerged adults were blood-fed. The eggs obtained from them were hatched in either water from the field or in dechlorinated tap water. The mosquito colonies were maintained for 10 generations. Midguts of female adults from unfed F₀ (emerging from field-caught pupae and larvae), F₅ and F₁₀ were dissected out and genomic DNA was extracted for 16S metagenomic sequencing. The sequences were compared to investigate the diversity and bacterial compositional differences using ANCOM and correlation clustering methods.

Results: Less than 10% of the bacterial families identified had differential relative abundances between generational groups and accounted for 46% of the variation observed. Although diversity reduced in F₁₀ mosquitoes during laboratory colonization (Shannon-Weaver; P-value < 0.05), 50% of bacterial genera were conserved in those bred continuously in field-water compared to 38% in those bred in dechlorinated tap water.

Conclusions: To our knowledge, this study is the first report on the assessment of gut bacterial community of mosquitoes during laboratory colonization and recommends the use of water from the natural breeding habitats if they are intended for microbiota research.

Keywords: Anopheles gambiae (sensu lato); Breeding habitat; Field water; Laboratory colonization; Midgut microbiota.

Fig. 1

Shannon-Weaver (a), Faith’s phylogenetic piversity (PD) (b) and Pielou’s evenness indices (c) for samples. Black dots indicate index measure for each sample and error bars show standard error of the mean for each treatment replicate. Red dotted lines are the total average for all replicates for an experimental group

Fig. 2

Comparison of observed OTUs between experimental treatments. Points are OTUs from samples (pool of 5 midguts) of each treatment. Error bars represent standard error of the mean

Fig. 3

Comparison of 99 taxonomically identified bacterial families between experimental groups. Each point represents a sample (pool of 5 midguts). a Comparison between the baseline and all field and lab water bred samples. b Comparison of baseline and generational groups of field-water-bred mosquitoes. Error bars represent standard error of the mean

Fig. 4

Comparison of 9 bacterial families (relative abundance ≥ 1% of sequences) between experimental groups. Points represent samples. Error bars represent standard error of the mean

Fig. 5

Volcano plot for the analysis of composition of microbiomes (ANCOM) test. Only significant bacterial taxa are labelled. Taxa on the top-left corner are distinct species but with small proportions, i.e. low f-score. Truly different taxa are depicted as one moves towards the far right (high W-statistic)

Fig. 6

Dendogram of bacterial families resulting from unsupervised correlation clustering. Balances (y0-y9) are shown by pink (numerator) and red (denominator) vertical bars on the left side of the map, and are not related to the scale