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Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant,  Solenopsis invicta  Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S.  S .  invicta  populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182  S .  invicta  colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove  S .  invicta  genes, known  S .  invicta  virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled  de novo  yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family  Dicistroviridae . Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that  S .  invicta  serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with  S .  invicta . This study expands knowledge of the RNA virome diversity found within  S .  invicta  populations.

Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina