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. 2014 Aug 24:7:391.
doi: 10.1186/1756-3305-7-391.

Comparative assessment of the bacterial communities associated with Aedes aegypti larvae and water from domestic water storage containers

Nsa Dada  1 , Estelle Jumas-Bilak, Sylvie Manguin, Razak Seidu, Thor-Axel Stenström, Hans J Overgaard
Affiliations

Comparative assessment of the bacterial communities associated with Aedes aegypti larvae and water from domestic water storage containers

Nsa Dada et al. Parasit Vectors. .

Abstract

Background: Domestic water storage containers constitute major Aedes aegypti breeding sites. We present for the first time a comparative analysis of the bacterial communities associated with Ae. aegypti larvae and water from domestic water containers.

Methods: The 16S rRNA-temporal temperature gradient gel electrophoresis (TTGE) was used to identify and compare bacterial communities in fourth-instar Ae. aegypti larvae and water from larvae positive and negative domestic containers in a rural village in northeastern Thailand. Water samples were cultured for enteric bacteria in addition to TTGE. Sequences obtained from TTGE and bacterial cultures were clustered into operational taxonomic units (OTUs) for analyses.

Results: Significantly lower OTU abundance was found in fourth-instar Ae. aegypti larvae compared to mosquito positive water samples. There was no significant difference in OTU abundance between larvae and mosquito negative water samples or between mosquito positive and negative water samples. Larval samples had significantly different OTU diversity compared to mosquito positive and negative water samples, with no significant difference between mosquito positive and negative water samples. The TTGE identified 24 bacterial taxa, belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and TM7 (candidate phylum). Seven of these taxa were identified in larval samples, 16 in mosquito positive and 13 in mosquito negative water samples. Only two taxa, belonging to the phyla Firmicutes and Actinobacteria, were common to both larvae and water samples. Bacilli was the most abundant bacterial class identified from Ae. aegypti larvae, Gammaproteobacteria from mosquito positive water samples, and Flavobacteria from mosquito negative water samples. Enteric bacteria belonging to the class Gammaproteobacteria were sparsely represented in TTGE, but were isolated from both mosquito positive and negative water samples by selective culture.

Conclusions: Few bacteria from water samples were identified in fourth-instar Ae. aegypti larvae, suggesting that established larval bacteria, most likely acquired at earlier stages of development, control the larval microbiota. Further studies at all larval stages are needed to fully understand the dynamics involved. Isolation of enteric bacteria from water samples supports earlier outcomes of E. coli contamination in Ae. aegypti infested domestic containers, suggesting the need to further explore the role of enteric bacteria in Ae. aegypti infestation.

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Figures

Figure 1
Figure 1
Alpha diversity rarefaction curves of bacterial OTUs from TTGE. Alpha diversity rarefaction curves of bacterial OTUs from Ae. aegypti larvae, mosquito positive and mosquito negative water samples analyzed by TTGE. This is based on Shannon-Wiener index (A), Observed species index (B) and Faith’s phylogenetic diversity (C). Curves represent mean diversity indices for each sample, and error bars represent standard error of means.
Figure 2
Figure 2
Boxplots of unweighted UniFrac distances of bacterial OTUs from TTGE. Boxplots show distribution of unweighted Unifrac distances of bacterial operational taxonomic units (OTUs) within and between Ae. aegypti larvae, mosquito positive and mosquito negative water samples. Brackets show outcomes of two-sample t-test comparisons of unweighted UniFrac distances; **P < 0.01; ns, not significant.
Figure 3
Figure 3
PCoA of unweighted UniFrac distances between samples analyzed by TTGE. Principal coordinate analysis of unweighted UniFrac distances between Ae. aegypti larvae and water from mosquito positive and negative containers. Distinct clusters of larval samples (red) and mosquito positive water samples (blue) are captured on all three PCoA plots. Mosquito negative water samples (orange) do not show any distinct clusters on any of the plots. Each axis show percentage of variation explained. Each data point consists of a central point surrounded by ellipsoids that indicate variation in UniFrac distances from rarefaction. This demonstrates that the clustering pattern holds up to subsampling.
Figure 4
Figure 4
Relative abundance of bacterial taxa obtained from TTGE sequences. Bars show mean relative abundance of the different bacterial taxa isolated by TTGE from Ae. aegypti larvae, mosquito positive and mosquito negative water samples. **Sequences that were not classified by Qiime. ***Low abundance phyla that are automatically grouped together by QIIME.
Figure 5
Figure 5
Number of bacterial taxa obtained from TTGE sequences. Venn diagram showing number of bacterial taxa obtained from TTGE analysis of Ae. aegypti larvae, mosquito positive and mosquito negative water samples.
Figure 6
Figure 6
Relative abundance of bacterial taxa obtained from cultured water samples. Bars represent mean relative abundance of bacterial taxa isolated from mosquito positive and negative water samples cultured on drigalski agar.
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