Metatranscriptomics and pyrosequencing facilitate discovery of potential viral natural enemies of the invasive Caribbean crazy ant, Nylanderia pubens

Abstract

Background: Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens.

Methodology and principal findings: Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens.

Conclusions: Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest.

Figure 1. Stepwise decision tree employed as a guide to assess the likelihood that a given sequence was of RNA (A) or DNA (B) viral origin.

Figure 2. Diagram summarizing the work flow and results for expression library creation, sequencing, assembly and kingdom assignments for the Caribbean crazy ant, Nylanderia pubens (top left).

Figure 3. Distribution by phyla of N. pubens sequences with significant (expectation score ≤1e⁻⁴) BLAST identity (upper panel).

Sequences with arthropod identity were further categorized by order (left, lower panel). Lastly, sequences with hymenoptera identity were further categorized by family (right, lower panel). All values are shown as percentages.

Figure 4. N. pubens sequences annotated with a gene ontology term (GO) and compared with reference sequences of Homo sapiens, Drosophila melanogaster, and Formicidae.