RNA shotgun metagenomic sequencing of northern California (USA) mosquitoes uncovers viruses, bacteria, and fungi

Abstract

Mosquitoes, most often recognized for the microbial agents of disease they may carry, harbor diverse microbial communities that include viruses, bacteria, and fungi, collectively called the microbiota. The composition of the microbiota can directly and indirectly affect disease transmission through microbial interactions that could be revealed by its characterization in natural populations of mosquitoes. Furthermore, the use of shotgun metagenomic sequencing (SMS) approaches could allow the discovery of unknown members of the microbiota. In this study, we use RNA SMS to characterize the microbiota of seven individual mosquitoes (species include Culex pipiens, Culiseta incidens, and Ochlerotatus sierrensis) collected from a variety of habitats in California, USA. Sequencing was performed on the Illumina HiSeq platform and the resulting sequences were quality-checked and assembled into contigs using the A5 pipeline. Sequences related to single stranded RNA viruses of the Bunyaviridae and Rhabdoviridae were uncovered, along with an unclassified genus of double-stranded RNA viruses. Phylogenetic analysis finds that in all three cases, the closest relatives of the identified viral sequences are other mosquito-associated viruses, suggesting widespread host-group specificity among disparate viral taxa. Interestingly, we identified a Narnavirus of fungi, also reported elsewhere in mosquitoes, that potentially demonstrates a nested host-parasite association between virus, fungi, and mosquito. Sequences related to 8 bacterial families and 13 fungal families were found across the seven samples. Bacillus and Escherichia/Shigella were identified in all samples and Wolbachia was identified in all Cx. pipiens samples, while no single fungal genus was found in more than two samples. This study exemplifies the utility of RNA SMS in the characterization of the natural microbiota of mosquitoes and, in particular, the value of identifying all microbes associated with a specific host.

Keywords: Bunyaviridae; Culex pipiens; Culiseta; Ochlerotatus; Rhabdoviridae; metagenomics; microbiota; shotgun sequencing.

FIGURE 1

Collection locations and a summary of the microbial sequences identified in each sample. Locations A through G correspond to the samples as listed in Table 1. A summary of the viral, bacterial, and fungal sequences identified in each sample is included. Details for each microbial group can be found in Tables 2–4. The map was created using Google Earth Pro, with data from the U.S. Navy, LDEO-Columbia, NSF, NOAA, SIO, NGA, MBARI, and GEBCO.

FIGURE 2

Phylogenetic history of all genera in the Bunyaviridae including sequences from this study. This consensus phylogeny was generated in MrBayes (Ronquist and Huelsenbeck, 2003) based on two independent chains run for one million generations to convergence. The average standard deviation of split frequencies was 0.000008. Posterior node probabilities are shown at nodes. Branch lengths are scaled to substitutions/site. Viral sequences uncovered in this study are labeled as such. Closely related mosquito-associated viruses are marked with an asterisk. The accession numbers of all sequences are available at http://dx.doi.org/10.6084/m9.figshare.1247641

FIGURE 3

Phylogenetic history of the Rhabdoviruses including sequences from this study. This consensus phylogeny was generated in MrBayes (Ronquist and Huelsenbeck, 2003) based on two independent chains run for one million generations to convergence. The average standard deviation of split frequencies was 0.002810. Posterior node probabilities are shown at nodes. Branch lengths are scaled to substitutions/site. Viral sequences uncovered in this study are labeled as such. Closely related mosquito-associated viruses are marked with an asterisk. The accession numbers of all sequences are available at http://dx.doi.org/10.6084/m9.figshare.1247641.

FIGURE 4

Phylogenetic history of select dsRNA viruses including sequences from this study and Chandler et al. (2014). This consensus phylogeny was generated in MrBayes (Ronquist and Huelsenbeck, 2003) based on two independent chains run for one million generations to convergence. The average standard deviation of split frequencies was 0.000948. Posterior node probabilities are shown at nodes. Branch lengths are scaled to substitutions/site. Viral sequences uncovered in this study are labeled as such. Closely related mosquito-associated viruses are marked with an asterisk. As in Ito et al. (2013), Penicillium chrysogenum virus is designated the outgroup. Proposed genera (Ito et al., 2013) are designated NG1, NG2, and NG3. The accession numbers of all sequences are available at http://dx.doi.org/10.6084/m9.figshare.1247641.

FIGURE 5

Phylogenetic history of the Narnaviruses including sequences from this study. This consensus phylogeny was generated in MrBayes (Ronquist and Huelsenbeck, 2003) based on two independent chains run for one million generations to convergence. Ourmiaviruses, which share the RNA-dependent RNA polymerase through a genomic rearrangement, are included as the outgroup. The average standard deviation of split frequencies was 0.004048. Posterior node probabilities are shown at nodes. Branch lengths are scaled to substitutions/site. Viral sequences uncovered in this study are labeled as such. Closely related mosquito-associated viruses are marked with an asterisk. Sequences labeled TSA were uncovered in the NCBI Transcriptome Shotgun Assembly database (Cook et al., 2013). The accession numbers of all sequences are available at .