Prevalence and Genetic Diversity of a Microsporidian Parasite in the Black Imported Fire Ant and Its Social Parasitic Ant (Formicidae: Myrmicinae: Solenopsis) in Buenos Aires Province, Argentina.

Microsporidia are natural pathogens of arthropods and have been used as biological control against insect pests. In the United States, efforts to control the invasive Red Imported Fire Ant, Solenopsis invicta, and Black Imported Fire Ant, Solenopsis richteri, have included the use of the microsporidium, Kneallhazia solenopsae. However, there is limited information about the genetic differences among the microsporidian variants found in S. invicta and in S. richteri. In this study, we assessed the prevalence and genetic diversity of K. solenopsae in native populations of S. richteri in Argentina (South America). Additionally, we examined the social parasitic ant, Solenopsis daguerrei, which is found in some S. richteri nests, for the presence of this microsporidium. The survey of 219 S. richteri nests revealed K. solenopsae infections in all five sites analyzed, with 28 colonies (12.8%) positive for the microsporidium. Among the 180 S. daguerrei individuals collected, seven ants (3.9%) from three sites tested positive for K. solenopsae. Phylogenetic analyses of the microsporidian variants present in S. richteri and S. daguerrei based on partial small subunit ribosomal gene sequences (SSU rRNA) showed that both ant species shared the same variant, which is different from the ones found in S. invicta. Further studies are needed to determine the pathogenicity of genetically different K. solenopsae variants among Solenopsis species.

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.

Effect of Irrigation on the Control of Red Imported Fire Ants (Hymenoptera: Formicidae) by Water-Resistant and Standard Fire Ant Baits.

The red imported fire ant, Solenopsis invicta (Buren), is an invasive pest of agricultural, urban, and natural areas. It is also considered a public health pest due to its painful stings. While it can be efficiently controlled by commercially available fire ant baits formulated with a corn-grit carrier, rain or irrigation is thought to degrade the carrier, compromising bait effectiveness. This study assessed the effect of irrigation on the efficacy of water-resistant and standard fire ant bait formulations, by comparing worker number, brood volume, and queen survivorship after access to water-soaked baits and to irrigated, bait-treated sod. In initial testing, wetted water-resistant and standard baits reduced fire ant colonies less than dry baits, both when baits were given to colonies directly and when colonies were given access to baits broadcasted (i.e., scattered) atop sod. Comparisons of the efficacy of piled versus broadcast applications of water-resistant and standard baits revealed reductions of >88% in adults and brood and no surviving queens for all bait treatments. This result was unexpected because piled baits were hypothesized to be better protected from irrigation than broadcast bait applications. In a field study, irrigated water-resistant and standard baits caused similar and significantly higher reductions in fire ant foraging activity relative to an untreated control. These results indicated that both the water-resistant and standard fire ant bait provided significant fire ant reductions even after irrigation.

Naturally Occurring Compounds/Materials as Alternatives to Synthetic Chemical Insecticides for Use in Fire Ant Management.

The invasive red imported fire ant, Solenopsis invicta Buren (hereafter, fire ants), is a significant threat to public health and a danger to livestock, pets and wildlife due to their venomous stings. The fire ant has invaded many countries and regions and has become a globally significant pest. The current major tool to manage fire ants are synthetic insecticides that are used largely as stomach poisons in bait products or contact insecticides in spray, broadcast, drench, and dust products for area and nest treatments. In addition to these insecticide products, repellants and fumigants can also be useful in some unique scenarios. The ever-increasing public concern about the potential adverse effects of synthetic insecticides on health and the environment has been a driving force for searching for safer alternatives to control fire ants. Tremendous effort has been made in developing biologically-based control for managing fire ants; however, natural products continue to be one of the most attractive sources of safe alternatives to synthetic insecticides. Here, we summarized the synthetic insecticides that are currently used in managing fire ants, available alternative products in the current market, and academic efforts in searching for fire ant natural toxins, repellants and fumigants.

Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva.

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species.

Evidence of niche shift and global invasion potential of the Tawny Crazy ant, Nylanderia fulva.

Analysis of an invasive species' niche shift between native and introduced ranges, along with potential distribution maps, can provide valuable information about its invasive potential. The tawny crazy ant, Nylanderia fulva, is a rapidly emerging and economically important invasive species in the southern United States. It is originally from east-central South America and has also invaded Colombia and the Caribbean Islands. Our objectives were to generate a global potential distribution map for N. fulva, identify important climatic drivers associated with its current distribution, and test whether N. fulva's realized climatic niche has shifted across its invasive range. We used MaxEnt niche model to map the potential distribution of N. fulva using its native and invaded range occurrences and climatic variables. We used principal component analysis methods for investigating potential shifts in the realized climatic niche of N. fulva during invasion. We found strong evidence for a shift in the realized climatic niche of N. fulva across its invasive range. Our models predicted potentially suitable habitat for N. fulva in the United States and other parts of the world. Our analyses suggest that the majority of observed occurrences of N. fulva in the United States represent stabilizing populations. Mean diurnal range in temperature, degree days at ≥10°C, and precipitation of driest quarter were the most important variables associated with N. fulva distribution. The climatic niche expansion demonstrated in our study may suggest significant plasticity in the ability of N. fulva to survive in areas with diverse temperature ranges shown by its tolerance for environmental conditions in the southern United States, Caribbean Islands, and Colombia. The risk maps produced in this study can be useful in preventing N. fulva's future spread, and in managing and monitoring currently infested areas.

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.

Mitigating the allergic effects of fire ant envenomation with biologically based population reduction.

PURPOSE OF REVIEW: To describe the current efforts to use biological control agents to reduce fire ant population levels, thus ultimately reducing the number of human sting and allergic reaction incidents. RECENT FINDINGS: Climate change and worldwide fire ant expansion will increase the frequency of human encounters and allergenic events, putting additional pressure on the public health sector. Six species of fire ant decapitating flies are now established in the United States. The microsporidium Kneallhazia solenopsae is well established and new fire ant hosts have been identified. The fire ant virus Solenopsis invicta virus 3 shows good potential for use as an environmentally friendly biopesticide because of its virulence and host specificity. SUMMARY: During separate founding events in the United States, Australia, mainland China, and Taiwan, fire ants native to South America escaped their native pathogens and parasites. Consequently, fire ant populations in these introduced regions pose a serious public health threat to the human populations by envenomation and subsequent allergic reactions. Specific, self-sustaining biological control agents have been discovered, studied, and released into fire ant populations in the United States in an effort to re-establish an ecological/competitive balance, resulting in reduced fire ant densities and human exposure.

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.

Multilocus genetic characterization of two ant vectors (Group II "Dirty 22" species) known to contaminate food and food products and spread foodborne pathogens.

The U.S. Food and Drug Administration utilizes the presence of filth and extraneous materials as one of the criteria for implementing regulatory actions and assessing adulteration of food products of public health importance. Twenty-two prevalent pest species (also known as the ''Dirty 22'' species) have been considered by this agency as possible vehicles for the spread of foodborne diseases, and the presence of these species is considered an indicator of unsanitary conditions in food processing and storage facilities. In a previous study, we further categorized the Dirty 22 species into four groups: group I includes four cockroach species, group II includes two ant species, group III includes 12 fly species, and group IV includes four rodent species. Here, we describe the development of three nested PCR primer sets and multilocus genetic characterization by amplifying the small subunit rRNA, elongation factor 1-alpha, and wingless (WNT-1) genes of group II Dirty 22 ant species Monomorium pharaonis and Solenopsis molesta. These novel group II Dirty 22 species-specific nested PCR primer sets can be used when the specimens cannot be identified using conventional microscopic methods. These newly developed assays will provide correct identification of group II Dirty 22 ant species, and the information can be used in the control of foodborne pathogens.

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

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.

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.

Molecular diversity of the microsporidium Kneallhazia solenopsae reveals an expanded host range among fire ants in North America.

Kneallhazia solenopsae is a pathogenic microsporidium that infects the fire ants Solenopsis invicta and Solenopsis richteri in South America and the USA. In this study, we analyzed the prevalence and molecular diversity of K. solenopsae in fire ants from North and South America. We report the first empirical evidence of K. solenopsae infections in the tropical fire ant, Solenopsis geminata, and S. geminata×Solenopsis xyloni hybrids, revealing an expanded host range for this microsporidium. We also analyzed the molecular diversity at the 16S ribosomal RNA gene in K. solenopsae from the ant hosts S.invicta, S. richteri, S. geminata and S. geminata×S. xyloni hybrids from North America, Argentina and Brazil. We found 22 16S haplotypes. One of these haplotypes (WD_1) appears to be widely distributed, and is found in S. invicta from the USA and S. geminata from southern Mexico. Phylogenetic analyses of 16S sequences revealed that K. solenopsae haplotypes fall into one of two major clades that are differentiated by 2-3%. In some cases, multiple K. solenopsae haplotypes per colony were found, suggesting either an incomplete homogenization among gene copies within the 16S gene cluster or multiple K. solenopsae variants simultaneously infecting host colonies.

Exploitation of a high genomic mutation rate in Solenopsis invicta virus 1 to infer demographic information about its host, Solenopsis invicta.

The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan in an attempt to infer demographic information about the recent S. invicta introduction into Taiwan by phylogenetic analysis. Nucleotide sequences were calculated to exhibit an overall identity of >90% between geographically-separated samples. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. Examination of the amino acid alignment for the RdRp revealed sequence identity >98% with only nine amino acid changes observed. Seven of these changes occurred in less than 4.3% of samples, while 2 (at positions 1266 and 1285) were featured prominently. Changes at positions 1266 and 1285 accounted for 36.2% and 34.0% of the samples, respectively. Two distinct groups were observed based on the amino acid residue at position 1266, Threonine or Serine. In cases where this amino acid was a Threonine, 90% of these sequences possessed a corresponding Valine at position 1285; only 10% of the Threonine(1266)-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine(1266) group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine(1266)/Valine(1285) and Serine(1266)/Isoleucine(1285) combinations are predominant phenotypes.

Expressed sequence tags from the red imported fire ant, Solenopsis invicta: annotation and utilization for discovery of viruses.

An expression library was created and 2304 clones sequenced from a monogyne colony of Solenopsis invicta. The primary intention of the project was to utilize homologous gene identification to facilitate discovery of viruses infecting this ant pest that could potentially be used in pest management. Additional genes were identified from the ant host and associated pathogens that serve as an important resource for studying these organisms. After assembly and removal of mitochondrial and poor quality sequences, 1054 unique sequences were yielded and deposited into the GenBank database under Accession Nos. EH412746 through EH413799. At least nine expressed sequence tags (ESTs) were identified as possessing microsatellite motifs and 15 ESTs exhibited significant homology with microsporidian genes. These sequences most likely originated from Thelohania solenopsae, a well-characterized microsporidian that infects S. invicta. Six ESTs exhibited significant homology with single-stranded RNA viruses (3B4, 3F6, 11F1, 12G12, 14D5, and 24C10). Subsequent analysis of these putative viral ESTs revealed that 3B4 was most likely a ribosomal gene of S. invicta, 11F1 was a single-stranded RNA (ssRNA) virus contaminant introduced into the colony from the cricket food source, 12G12 appeared to be a plant-infecting tenuivirus also introduced into the colony as a field contaminant, and 3F6, 14D5, and 24C10 were all from a unique ssRNA virus found to infect S. invicta. The sequencing project illustrates the utility of this method for discovery of viruses and pathogens that may otherwise go undiscovered.

Phenology, distribution, and host specificity of Solenopsis invicta virus-1.

Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p<0.0005, r=0.82) and SINV-1A (p<0.0001, r=0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV-1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Florida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV-1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S.