Effect of Solenopsis invicta virus 3 on brood mortality and egg hatch in Solenopsis invicta.

Solenopsis invicta virus 3 (SINV-3) has been shown to cause significant mortality among all stages of its host, Solenopsis invicta. One impact of the virus is alteration of worker ant foraging behavior, which results in colony starvation and collapse over time. Additionally, it has been hypothesized that SINV-3 infection of S. invicta may disrupt worker ant brood care behavior. To investigate this possibility, various combinations of SINV-3-infected and -uninfected adult (worker) and immature (brood) stages were placed together and monitored using the response variables, mortality, egg hatch, and virus load. While significant differences in percent cumulative S. invicta worker ant mortality among six combinations of SINV-3-infected and -uninfected stages were observed, no significant differences in percent cumulative mortality of S. invicta larvae or pupae were observed. No significant differences in egg hatch were observed among SINV-3-uninfected, SINV-3-infected (colony-treated and queen-treated), and starved colonies. Eggs hatched normally in 10-12 days for all treatments indicating that egg care by worker ants was unaffected by SINV-3 infection status. The study further clarifies SINV-3 pathogenesis in its host, S. invicta. Larval mortality in SINV-3-infected colonies does not appear to be caused by worker ant neglect. S. invicta brood under the care of SINV-3-infected worker ants did not exhibit higher mortality rates compared with those tended by SINV-3-uninfected worker ants.

RNA virus discoveries in the electric ant, Wasmannia auropunctata.

Despite being one of the most destructive invasive species of ants, only two natural enemies are known currently for Wasmannia auropunctata, commonly known as the electric ant or little fire ant. Because viruses can be effective biological control agents against many insect pests, including ants, a metagenomics/next-generation sequencing approach was used to facilitate discovery of virus sequences from the transcriptomes of W. auropunctata. Five new and complete positive sense, single-stranded RNA virus genomes, and one new negative sense, single-stranded RNA virus genome were identified, sequenced, and characterized from W. auropunctata collected in Argentina by this approach, including a dicistrovirus (Electric ant dicistrovirus), two polycipiviruses (Electric ant polycipivirus 1; Electric ant polycipivirus 2), a solinvivirus (Electric ant solinvivirus), a divergent genome with similarity to an unclassified group in the Picornavirales (Electric ant virus 1), and a rhabdovirus (Electric ant rhabdovirus). An additional virus genome was detected that is likely Solenopsis invicta virus 10 (MH727527). The virus genome sequences were absent from the transcriptomes of W. auropunctata collected in the USA (Hawaii and Florida). Additional limited field surveys corroborated the absence of these viruses in regions where the electric ant is invasive (the USA and Australia). The replicative genome strand of four of the viruses (Electric ant polycipivirus 2, Electric ant solinvivirus, Electric ant virus 1, and Solenopsis invicta virus 10 (in the electric ant) was detected in Argentinean-collected W. auropunctata indicating that the ant is a host for these viruses. These are the first virus discoveries to be made from W. auropunctata.

Characterization of Solenopsis invicta virus 4, a polycipivirus infecting the red imported fire ant Solenopsis invicta.

Solenopsis invicta virus 4 (SINV-4), a new polycipivirus, was characterized in the host in which it was discovered, Solenopsis invicta. SINV-4 was detected in the worker and larval stages of S. invicta, but not in pupae, male or female alates, or queens. The SINV-4 titer was highest in worker ants, with a mean of 1.14 × 107 ± 5.84 ×107 SINV-4 genome equivalents/ng RNA. Electron microscopic examination of negatively stained samples from particles purified from SINV-4-infected fire ant workers revealed isometric particles with a mean diameter of 47.3 ± 1.4 nm. The mean inter-colony SINV-4 infection rate among S. invicta worker ants was 45.8 ± 38.6 in Alachua County, Florida. In S. invicta collected in Argentina, SINV-4 was detected in 22% of 54 colonies surveyed from across the Formosa region. There did not appear to be any seasonality associated with the SINV-4 infection rate among S. invicta nests. SINV-4 was successfully transmitted to uninfected S. invicta colonies by feeding. Among three colonies of S. invicta inoculated with SINV-4, two retained the infection for up to 72 days. The replicative genome strand of SINV-4 was detected in 18% (n = 11) of SINV-4-infected S. invicta colonies. Among 33 ant species examined, the plus genome strand of SINV-4 was detected in undetermined species of Dorymyrmex and Pheidole, Cyphomyrmex rimosus, Monomorium pharaonis, Pheidole obscurithorax, Solenopsis geminata, Solenopsis richteri, Solenopsis xyloni, and Solenopsis invicta. However, the replicative (minus) genome strand was only detected in S. invicta. SINV-4 infection did not impact brood production or queen fecundity in S. invicta. The mean brood rating (63.3% ± 8.8) after 31 days for SINV-4-infected colonies was not statistically different from that of uninfected colonies (48.3 ± 25.5). At the end of the 31-day test period, mean egg production was not significantly different between SINV-4-infected S. invicta colonies (287.7 ± 45.2 eggs laid/24 hours) and uninfected control colonies (193.0 ± 43.6 eggs laid/24 hours).

Field evaluation of Solenopsis invicta virus 3 against its host Solenopsis invicta.

Viruses have been used successfully as biocontrol agents against several insect pests but not ants. Laboratory tests have shown that Solenopsis invicta virus 3 (SINV-3) may be an effective natural control agent against its host, the red imported fire ant (Solenopsis invicta Buren). In this field trial, SINV-3 was released into 12 active S. invicta nests within a 0.088-hectare area in Florida and the impact on the ants monitored. SINV-3 was successfully transmitted, established, and multiplied within treated colonies reaching a maximum mean value of 8.71 × 108 ± 8.26 × 108 SINV-3 genome equivalents/worker ant 77 days after inoculation. SINV-3 was not detected in any of the nests in the control group. A 7-fold decrease in nests was observed in the SINV-3-treated group compared with the untreated control. A correspondingly significant decrease in S. invicta nest size also was observed over the course of the evaluation. Based on the nest rating scale, nest size among those treated with SINV-3 decreased from 3.92 ± 1.24 on day 0 to 1.67 ± 2.06 on day 77, which represents a 57.4% decrease in size. Conversely, the nest rating for the control group increased 9.3%, from 4.42 ± 1.24 on day 0 to 4.83 ± 2.12 on day 77. A follow-up survey of SINV-3-treated and -untreated plots conducted 9 months after initial treatment revealed that fire ant populations rebounded, but at a different rate. A total of 11 and 19 nests were detected in the SINV-3-treated and -untreated areas, respectively. SINV-3 was still detected in the treated area 1.8 years after the initial virus treatment and the virus had spread into the adjacent control plot. Results demonstrate that SINV-3 is an effective natural control agent against the invasive ant, S. invicta; the virus causes no known detrimental ecological impacts, is host specific, and sustained in the environment.

ICTV Virus Taxonomy Profile: Solinviviridae.

Solinviviridae is a family of picorna/calici-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-11 kb. Unusually, their capsid proteins are encoded towards the 3'-end of the genome where they can be expressed both from a subgenomic RNA and as an extension of the replication (picorna-like helicase-protease-polymerase) polyprotein. Members of two species within the family infect ants, but related unclassified virus sequences derive from a large variety of insects and other arthropods. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Solinviviridae, which is available at www.ictv.global/report/solinviviridae.

ICTV Virus Taxonomy Profile: Polycipiviridae.

Polycipiviridae is a family of picorna-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-12 kb. Unusually for viruses within the order Picornavirales, their genomes are polycistronic, with four (or more) consecutive 5'-proximal open reading frames (ORFs) encoding structural (and possibly other) proteins and a long 3' ORF encoding the replication polyprotein. Members of species within the family have all been detected in ants or via arthropod transcriptomic datasets. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Polycipiviridae, which is available at www.ictv.global/report/polycipiviridae.

Nine new RNA viruses associated with the fire ant Solenopsis invicta from its native range.

The red imported fire ant (Solenopsis invicta) escaped its natural enemies when it was introduced into North America in the 1930s from South America. US efforts have focused on discovery of natural enemies, like viruses, to provide sustainable control of the ant. Nine new virus genomes were sequenced from the invasive fire ant Solenopsis invicta using metagenomic RNA sequencing. The virus genomes were verified by Sanger sequencing and random amplification of cDNA ends reactions. In addition to the nine new virus genomes, the previously described Solenopsis viruses were also detected, including Solenopsis invicta virus 1 (SINV-1), SINV-2, SINV-3, SINV-4, SINV-5, and Solenopsis invicta densovirus. The virus sequences came from S. invicta workers, larvae, pupae, and dead workers taken from midden piles collected from across the ant's native range in Formosa, Argentina. One of the new virus genomes (Solenopsis invicta virus 6) was also detected in populations of North American S. invicta. Phylogenetic analysis of the RNA dependent RNA polymerase, the entire nonstructural polyprotein, and genome characteristics were used to tentatively taxonomically place these new virus genome sequences; these include four new species of Dicistroviridae, one Polycipiviridae, one Iflaviridae, one Totiviridae, and two genome sequences that were too taxonomically divergent to be placed with certainty. The S. invicta virome is the best characterized from any ant species and includes 13 positive-sense, single-stranded RNA viruses (Solenopsis invicta virus 1 to Solenopsis invicta virus 13), one double-stranded RNA virus (Solenopsis midden virus), and one double-stranded DNA virus (Solenopsis invicta densovirus). These new additions to the S. invicta virome offer potentially new classical biological control agents for S. invicta.

Influence of temperature on the pathogenicity of Solenopsis invicta virus 3.

Field evaluations assessing the prevalence of Solenopsis invicta virus 3 (SINV-3) have shown that the virus exhibits a distinct seasonal phenology in the host, Solenopsis invicta, that is negatively correlated with warmer temperatures. Active SINV-3 infections were established in Solenopsis invicta colonies, which were subsequently maintained at 19.1, 22.2, 25.5, 27.7, and 29.3 °C. The quantity of brood declined in all SINV-3-treated colonies regardless of temperature over the initial 30 days. However, the quantity of brood in colonies held at 29.3 °C began increasing (recovering) in the next 40 days until they were statistically equivalent to untreated control colonies. Meanwhile, the quantity of brood continued to decline in colonies held at 19.1, 22.2, 25.5, and 27.7 °C for the duration of the test (81days). By the end of the test, these colonies were in poor health as indicated by decreased brood. Conversely, the amount of brood for colonies held at 29.3 °C increased to above 3, indicating healthy vigorous growth. Worker ants from SINV-3-treated colonies maintained at 19.1, 22.2, and 25.5 °C showed strong production of the VP2 capsid protein by Western blotting; 100% of the colonies sampled (n = 3) showed production of VP2. However, VP2 was detected in only 33% of colonies maintained at 27.7 °C, and the VP2 response was nearly undetectable in all colonies maintained at 29.3 °C. These results indicate that virus assembly does not appear to be occurring efficiently at the higher temperatures. Also, the quantity of SINV-3 detected in queens was significantly lower in those maintained at 29.3 °C compared with the lower temperature treatments. These results indicate that warm summer temperatures combined with fire ant thermoregulatory behavior and perhaps behavioral fevers may explain the low prevalence of SINV-3 in fire ant colonies during the summer.

Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses.

Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5' third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3' two-thirds of the genome. The polypeptide translation of this 3' open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. An inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and in silico assembly of RNA isolated from Solenopsis invicta and four other ant species, followed by targeted Sanger sequencing, we obtained nearly complete genomes for four further viruses in the group. Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent (pairwise amino acid identities of as low as 17 % in the non-structural polyprotein), but possess the same overall polycistronic genome structure, which is distinct from all other characterized picorna-like viruses. Consequently, we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species) and Sopolycivirus (11 species), with members of the latter infecting ants in at least 3 different subfamilies.

Development of a lateral flow immunoassay for rapid field detection of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

The red imported fire ant, Solenopsis invicta, is an aggressive, highly invasive pest ant species from South America that has been introduced into North America, Asia, and Australia. Quarantine efforts have been imposed in the USA to minimize further spread of the ant. To aid the quarantine efforts, there remains an acute need for a rapid, field portable method for the identification of these ants. In this report, we describe two novel monoclonal antibodies that specifically bind the S. invicta venom protein 2 produced by S. invicta. Using these monoclonal antibodies we developed a lateral flow immunoassay that provides a rapid and portable method for the identification of S. invicta ants. The lateral flow immunoassay was validated against purified S. invicta venom protein 2 and 33 unique ant species (representing 15 % of the total species and 42 % of the Myrmicinae genera found in Florida), and only S. invicta and the S. invicta/richteri hybrid produced a positive result. These monoclonal antibodies were selective to S. invicta venom protein 2 and did not bind to proteins from congeners (i.e., S. geminata or S. richteri) known to produce a S. invicta venom protein 2 ortholog. This S. invicta lateral flow immunoassay provides a new tool for regulatory agencies in the USA to enforce quarantine protocols and limit the spread of this invasive ant. Graphical Abstract Field method to detect and identify the red imported fire ant, Solenopsis invicta.

Dose response of red imported fire ant colonies to Solenopsis invicta virus 3.

Baiting tests were conducted to evaluate the effect of increasing Solenopsis invicta virus 3 (SINV-3) dose on fire ant colonies. Actively growing early-stage fire ant (Solenopsis invicta Buren) laboratory colonies were pulse-exposed for 24 hours to six concentrations of SINV-3 (10(1), 10(3), 10(5), 10(7), 10(9) genome equivalents/µl) in 1 ml of a 10 % sucrose bait and monitored regularly for two months. SINV-3 concentration had a significant effect on colony health. Brood rating (proportion of brood to worker ants) began to depart from the control group at 19 days for the 10(9) concentration and 26 days for the 10(7) concentration. At 60 days, brood rating was significantly lower among colonies treated with 10(9), 10(7), and 10(5) SINV-3 concentrations. The intermediate concentration, 10(5), appeared to cause a chronic, low-level infection with one colony (n = 9) supporting virus replication. Newly synthesized virus was not detected in any fire ant colonies treated at the 10(1) concentration, indicating that active infections failed to be established at this level of exposure. The highest bait concentration chosen, 10(9), appeared most effective from a control aspect; mean colony brood rating at this concentration (1.1 ± 0.9 at the 60 day time point) indicated poor colony health with minimal brood production. No clear relationship was observed between the quantity of plus genome strand detected and brood rating. Conversely, there was a strong relationship between the presence of the replicative genome strand and declining brood rating, which may serve as a predictor of disease severity. Recommendations for field treatment levels to control fire ants with SINV-3 are discussed.

Myrmecomorba nylanderiae gen. et sp. nov., a microsporidian parasite of the tawny crazy ant Nylanderia fulva.

A new microsporidian genus and species, Myrmecomorba nylanderiae, is described from North American populations of the tawny crazy ant, Nylanderia fulva. This new species was found to be heterosporous producing several types of binucleate spores in both larval and adult stages and an abortive octosporoblastic sporogony in adult ants. While microsporidia are widespread arthropod parasites, this description represents only the fifth species described from an ant host. Molecular analysis indicated that this new taxon is phylogenetically closely allied to the microsporidian family Caudosporidae, a group known to parasitize aquatic black fly larvae. We report the presence of 3 spore types (Type 1 DK, Type 2 DK, and octospores) with infections found in all stages of host development and reproductive castes. This report documents the first pathogen infecting N. fulva, an invasive ant of considerable economic and ecological consequence.

Host specificity and colony impacts of the fire ant pathogen, Solenopsis invicta virus 3.

An understanding of host specificity is essential before pathogens can be used as biopesticides or self-sustaining biocontrol agents. In order to define the host range of the recently discovered Solenopsis invicta virus 3 (SINV-3), we exposed laboratory colonies of 19 species of ants in 14 genera and 4 subfamilies to this virus. Despite extreme exposure during these tests, active, replicating infections only occurred in Solenopsis invicta Buren and hybrid (S. invicta×S. richteri) fire ant colonies. The lack of infections in test Solenopsis geminata fire ants from the United States indicates that SINV-3 is restricted to the saevissima complex of South American fire ants, especially since replicating virus was also found in several field-collected samples of the black imported fire ant, Solenopsis richteri Forel. S. invicta colonies infected with SINV-3 declined dramatically with average brood reductions of 85% or more while colonies of other species exposed to virus remained uninfected and healthy. The combination of high virulence and high host specificity suggest that SINV-3 has the potential for use as either a biopesticide or a self-sustaining biocontrol agent.

Successful transmission of Solenopsis invicta virus 3 to Solenopsis invicta fire ant colonies in oil, sugar, and cricket bait formulations.

Tests were conducted to evaluate whether Solenopsis invicta virus 3 (SINV-3) could be delivered in various bait formulations to fire ant colonies and measure the corresponding colony health changes associated with virus infection in Solenopsis invicta. Three bait formulations (10% sugar solution, cricket paste, and soybean oil adsorbed to defatted corn grit) effectively transmitted SINV-3 infections to S. invicta colonies. Correspondingly, viral infection was shown to be detrimental to colony health and productivity. By day 32, all ant colonies exposed to a single 24h pulse treatment of SINV-3 became infected with the virus regardless of the bait formulation. However, the SINV-3 sugar and cricket bait-treated colonies became infected more rapidly than the oil-treated colonies. Sugar and cricket-treated colonies exhibited significant declines in their brood ratings compared with the untreated control and oil bait-treated colonies. Measures of colony health and productivity evaluated at the end of the study (day 47) showed a number of differences among the bait treatments and the control group. Statistically significant and similar patterns were exhibited among treatments for the quantity of live workers (lower), live brood (lower), total colony weight (lower), worker mortality (higher), proportion larvae (lower), and queen weight (lower). Significant changes were also observed in the number of eggs laid by queens (lower) and the corresponding ovary rating in SINV-3-treated colonies. The study provides the first successful demonstration of SINV-3 as a potential biopesticide against fire ants.

Geographic distribution suggests that Solenopsis invicta is the host of predilection for Solenopsis invicta virus 1.

Solenopsis invicta virus 1 (SINV-1) was found regularly and prevalently in S. invicta. In sampled locations where S. invicta and S. geminata are sympatric (specifically, Gainesville, FL and Travis, TX), SINV-1 was detected in S. geminata. Conversely, in areas in which S. geminata and S. invicta are allopatric, SINV-1 was not detected in S. geminata; these locations included north Australia (n=12), southern Mexico (n=107), Hawaii (n=48), Taiwan (n=12), and the Johnston Atoll (n=6). A similar relationship was observed for S. richteri. In areas in which S. invicta and S. richteri were sympatric, SINV-1 was detected in the S. richteri population, but in areas in which S. invicta and S. richteri were allopatric, SINV-1 was not detected. These occurrences suggest that S. invicta is the host of predilection, or preferred host for SINV-1, and that the congenerics, S. geminata and S. richteri serve as either accidental, reservoir, or transfer hosts. The minus genome strand of SINV-1 was detected in S. geminata and S. richteri indicating that these species may serve as functional hosts capable of supporting SINV-1 replication. SINV-1 was not detected in S. xyloni regardless of its proximity to S. invicta. These results suggest that SINV-1 may be an example of pathogen spillover or pollution.

Solenopsis invicta virus 3 infection alters foraging behavior in its host Solenopsisinvicta.

Solenopsis invicta is an invasive ant introduced into the United States in the early 1900s. Control efforts and damage caused by this ant exceed $8 billion annually. Solenopsis invicta virus 3 (SINV-3) is a positive-sense, single-stranded RNA virus (Solinviviridae) that is being used as a classical natural control agent for S. invicta. S. invicta colonies were exposed to purified preparations of SINV-3 to investigate the impact of the virus on the ant. Food retrieval behavior (i.e., foraging) by worker ants was significantly decreased, which led to mortality among all life stages. Queen fecundity and weight were also significantly decreased. The change in food retrieval was associated with the exhibition of an unusual behavior, whereby the remaining live ant workers wedged dead ant worker corpses into and on top of cricket carcasses (the laboratory colony food source). SINV-3 infection alters foraging behavior in S. invicta, which adversely impacts colony nutrition.

No evidence for translation of pog, a predicted overlapping gene of Solenopsis invicta virus 1.

An overlapping open reading frame (ORF) with a potential to encode a functional protein has been identified within the 3'-proximal ORF of Solenopsis invicta virus 1 (SINV-1) and three bee viruses. This ORF has been referred to as predicted overlapping gene (pog). Protein motif searches of POG revealed weak relationships precluding assignment of a potential function. Neither a transcript nor a protein encoded by the pog ORF has been detected. However, recently, a protein encoded by the corresponding +1 overlapping ORF (termed ORFx) in the Israeli acute paralysis virus (IAPV) was demonstrated by recombinant means as well as in IAPV-infected honey bees. The objective of our study was to attempt to provide empirical evidence for the presence of a pog-derived protein from SINV-1-infected fire ants. A number of different laboratory and field SINV-1-infected Solenopsis invicta preparations were examined by western blotting for the presence of a POG protein sequence. In every case, these preparations failed to yield any detectable bands when probed with a polyclonal antibody preparation raised to a portion of the pog predicted protein sequence. Although impossible to prove a negative result, proper controls used in these studies suggested that the pog ORF is not translated into a functional protein in SINV-1.

Non-enzymatic hydrolysis of RNA in workers of the ant Nylanderia pubens.

During preparation of total RNA from Nylanderia pubens (Forel) (Hymenoptera: Formicidae) workers for use in expression library construction, severe RNA degradation consistently occurred. This degradation was masked by spectrophotometric analysis but clearly evident by microfluidic-based assay. Although not specifically identified, the degrading entity was endogenous and localized to the abdomen (terminal abdominal segments) of adult ants. RNA degradation was not observed in preparations of larvae, non-melanized pupae, or eggs. Various RNase and protease inhibitors had no protective effect. However, the metal chelating agent ethylenediaminetetraacetic acid prevented RNA degradation and provides insight into the occurrence.

Examination of host genome for the presence of integrated fragments of Solenopsis invicta virus 1.

A series of oligonucleotide primer pairs covering the entire genome of Solenopsis invicta virus 1 (SINV-1) were used to probe the genome of its host, S. invicta, for integrated fragments of the viral genome. All of the oligonucleotide primer sets yielded amplicons of anticipated size from cDNA created from an RNA template from SINV-1. However, no corresponding amplification was observed when genomic DNA (from 32 colonies of S. invicta) was used as template for the PCR amplifications. Host DNA integrity was verified by amplification of an ant-specific gene, SiGSTS1. The representation of fire ant colonies included both social forms, monogyne and polygyne, and those infected and uninfected with SINV-1. Furthermore, no amplification was observed from genomic DNA from ant samples collected from Argentina or the US. Thus, it appears that SINV-1 genome integration, or a portion therein, has not likely occurred within the S. invicta host genome.

Multiple virus infections occur in individual polygyne and monogyne Solenopsis invicta ants.

Concurrent infections of Solenopsis invicta colonies with S. invicta virus 1 (SINV-1), SINV-2, and SINV-3 has been reported. However, whether individual ants were capable of supporting multiple virus infections simultaneously was not known, nor whether the social form of the colony (polygyne or monogyne) had an influence on the occurrence of multiple infection rates in individual ants. S. invicta field populations were sampled sequentially to establish whether multiple virus infections co-occurred in individual worker ants. In addition, the intra-colony virus infection rates were compared in monogyne and polygyne field colonies to determine whether social form played a role in the viral infection prevalence. All combinations of virus infection (SINV-1, SINV-2, or SINV-3 alone, SINV-1 & SINV-2, SINV-1 & SINV-3, SINV-2 & SINV-3, and SINV-1, SINV-2 & SINV-3) were detected in individual worker ants as well as queens in the field. Thus, individual S. invicta ants can be infected simultaneously with all combinations of the S. invicta viruses. Colony social form did have an influence on the intra-colony prevalence of multiple S. invicta virus infections. Polygyne colonies exhibited significantly greater intra- and inter-colony single and multiple virus infections compared with monogyne colonies.