Bacterial Communities Associated with <i>Culex</i> Mosquito Larvae and Two Emergent Aquatic Plants of Bioremediation Importance

<div><p>Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito (<i>Culex</i><i>tarsalis</i>), the submerged portions of two emergent macrophytes (California bulrush, <i>Schoenoplectus</i><i>californicus</i> and alkali bulrush, <i>Schoenoplectus</i><i>maritimus</i>), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus <i>Thorsellia</i> (<i>Enterobacteriaceae</i>), was the most abundant phylum recovered from <i>C</i><i>. tarsalis</i> larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, <i>Cyanobacteria</i>, Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within <i>C</i><i>. tarsalis</i> larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although <i>Thorsellia</i> spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.</p> </div

Alpha diversity measures.

<p>Alpha diversity measures based on PD_Whole tree of the bacterial communities from mosquito larvae, water column and leaves. Sequences from mosquito samples are significantly less diverse than sequences from water and plant samples. The x-axis for the phylogenetic diversity of <i>Bacteria</i> communities from leaf samples is offset by 100 sequences for better illustration.</p

Taxonomic profiling of mosquito-water-plant microbiome profiles.

<p>PCoA plots of weighted UniFrac distances of bacterial communities in mosquitoes, water and leaf samples from mesocosms containing the two bulrushes (alkali and California bulrushes) from the three sampling dates. Panel A shows the OTUs associated with that region on the plot, scaled based on sequence abundance. Panel B shows a PCoA plot based on three DNA sources (mosquitoes, water and plant leaves), Panel C recolors samples of Panel B to highlight two plant species, Panel D recolors the same samples based on the three sample dates.</p

Overlap of bacterial communities across habitats.

<p>Venn diagram illustrating overlapping of <i>Bacteria</i> OTUs and sequences between mosquito larvae and habitat (<i>C</i><i>. tarsalis</i> larvae; habitat = leaves of alkali and California bulrushes; water = water column samples). The first number represents the number of OTUs, while the number in parentheses represents the number of sequences.</p

Community similarity of OTU profiles representation.

<p>PCoA plots based on Bray-Curtis distances of OTU profiles mosquitoes, water and leaf samples from mesocosms containing the two bulrushes from the different sampling dates. Panel A shows points colored by DNA source. Panel B shows points colored by the plant present in the mesocosm. Panel C shows points colored by sampling date.</p

Family-level classification of bacterial communities in mosquitoes.

<p>Family-level classification of bacterial communities in <i>C</i><i>. tarsalis</i> larvae and their relative proportions.</p

Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries

<div><p>High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.</p></div

Percent coverage of pooled sequencing result relative to barcoded sequencing result.

<p>Each of the 73 clones was categorized into Clone Types A, B, C, or D by the number of end-tags obtained (one or two), whether the end-tag retrieved a contig from the pool, and the completeness of the retrieved pooled sequencing result relative to the reference barcoded sequencing result (full or partial coverage). Clone Type descriptions are given above.</p

Alignment identity between pooled sequencing result and barcoded sequencing result.

<p>For all 73 clones, end-tags were used to retrieve contigs from pooled sequencing results; retrieved contigs were aligned to the reference barcoded sequencing result, and clones were binned by percent identity.</p

Overlapping clones assemble into one contig.

<p>Three overlapping clones as revealed by barcoded sequencing (above) and pooled sequencing (below). Locations of end-tags are indicated by vertical dashed lines. White dashed boxes indicate gaps in the pooled sequencing data; black boxes indicate a contig. Lengths of all contigs are given.</p