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.     

Infection characteristics of Solenopsis invicta virus 2 in the red imported fire ant, Solenopsis invicta

Solenopsis invicta virus 2 (SINV-2) is the second virus identified from the fire ant, S. invicta Buren. SINV-2 is unique among positive-strand RNA viruses from insects by possessing four cistrons in a monopartite genome. Fire ant colonies testing positive for SINV-2 by RT-PCR did not exhibit any discernable symptoms. RT-PCR-based surveys for SINV-2 among 688 fire ant mounds in Alachua County, Florida, sampled during the period January, 2006 through December, 2007 showed that the prevalence of SINV-2 among nests ranged from 1.6% to 16.4%. Unlike S. invicta virus 1, no seasonal-associated prevalence was observed with regard to SINV-2 infection among fire ant colonies. No social form specificity was evident; SINV-2 was found in both monogyne and polygyne S. invicta ants. Real-time quantitative PCR experiments showed that SINV-2 genome equivalents per individual ant ranged from 1.9 × 10⁷ in. pupae to 4.3 × 10¹¹ in. inseminated queens. The SINV-2 infection was detected in all ant stages examined (eggs, larvae, pupae, workers, and queens). Tissue tropism studies indicated that the alimentary canal (specifically the midgut) is most likely the susceptible tissue. SINV-2 was successfully transmitted to uninfected S. invicta ants by feeding a partially purified homogenate of SINV-2-infected ants. The SINV-2 transmission rate ranged from 30% to 80%, and both positive (genomic) and negative (replicative) SINV-2 RNA strands accumulated in recipient ants over the course of the experiment. These results indicated that SINV-2 replicates within S. invicta.     

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.     

Interspecific competition between Solenopsis invicta and two native ant species, Pheidole fervens and Monomorium chinense

This study was designed to understand the effects of the interspecific competition between red imported fire ant, Solenopsis invicta Buren and two native ant species, Pheidole fervens Smith and Monomorium chinense Santschi, by conducting colony interference and individual confrontation tests under laboratory conditions. The colony interference test showed that both native ant species, owing to their numerical advantage, killed the Solenopsis invicta virus-1 (SINV-1)-infected or healthy queens of S. invicta. Significantly less time was required for M. chinense to kill all SINV-1-infected S. invicta compared with the time required to kill the healthy S. invicta. Compared with healthy S. invicta, SINV-1-infected S. invicta spent a longer time eliminating the P. fervens colonies. In confrontation tests, M. chinense killed a significantly higher number of infected S. invicta minors than they did healthy minors, but the number of S. invicta majors (either infected or healthy) killed was substantially less. This study found that the viral infection weakened the competitive ability of S. invicta and made them prone to be eliminated by M. chinense but not by P. fervens.     

Solenopsis invicta virus-1A (SINV-1A): distinct species or genotype of SINV-1?

We have cloned and sequenced a 2845 bp cDNA representing the 3'-end of either a new picorna-like virus species or genotype of Solenopsis invicta virus-1 (SINV-1). Analysis of the nucleotide sequence revealed 1 large open reading frame. The amino acid sequence of the translated open reading frame was most identical to structural proteins of SINV-1 (97%), followed by the Kashmir bee virus (KBV, 30%), and acute bee paralysis virus (ABPV, 29%). A PCR-based survey for SINV-1 and the new species or genotype (tentatively named S. invicta virus-1A, SINV-1A) using RNA extracts of S. invicta collected around Gainesville, Florida, revealed a mean colony infestation rate of 25% by SINV-1 and 55% by SINV-1A. Both SINV-1 and SINV-1A were found to co-infect 17.5% of the nests surveyed. Although the data preclude definitive species or genotype assignment, there is no doubt that SINV-1A is distinct from SINV-1, identifiable, and infects S. invicta. We provide a simple RT-PCR technique capable of discerning SINV-1 and SINV-1A infection of S. invicta.     

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.     

Detection and quantitation of Solenopsis invicta virus-2 genomic and intermediary replicating viral RNA in fire ant workers and larvae

Quantitative real-time PCR (QPCR) was used to quantify the genome of Solenopsis invicta virus-2 (SINV-2) from infected individual ants of S. invicta. Strand-specific cDNA synthesis oligonucleotide primers and RNase digestion after cDNA synthesis allowed quantification of plus (genomic) and minus (replicative) strands of the SINV-2 genome. Both strands were detected in adult workers and larval fire ants indicating that the virus was replicating within the ant. The differences between the genomic to replicative strand ranged from 199-fold in larvae to 479-fold in workers with an average ratio of 339:1.     

Evidence of intracolony transmission of Thelohania solenopsae (Microsporidia: Thelohaniidae) in red imported fire ants (Hymenoptera: Formicidae) and the first report of spores from pupae.

Red imported fire ant, Solenopsis invicta, colonies were infected horizontally by introducing live brood (mainly larvae and pupae) infected with Thelohania solenopsae. Live, infected brood introduced into uninfected colonies were adopted and raised to adulthood instead of being executed by the recipient colony. Introductions of infected larvae with uninfected pupae, which eclose into adult worker caste fire ants, resulted in an 80% infection rate of the inoculated colonies. Infections from introductions of infected pupae with uninfected larvae resulted in a 37.5% infection of inoculated colonies. Infections were also detected in 11.6 and 3.7% of the adult worker caste ants that eclosed from uninfected large larvae and pupae, respectively, that were held with infected adult workers. Microscopic examination of infected brood revealed sporoblasts and large numbers of spores of T. solenopsae in S. invicta pupae.     

Transmission of Vairimorpha invictae (Microsporidia: Burenellidae) infections between red imported fire ant (Hymenoptera: Formicidae) colonies

Red imported fire ant, Solenopsis invicta, colonies were successfully infected with the microsporidium Vairimorpha invictae by introducing live larvae, pupae, or dead adults from V. invictae-infected field colonies collected in Argentina. Introductions with 4th instar larvae or non-melanized pupae obtained from infected field colonies, resulted in infection of 40% of the inoculated colonies. Introductions of 4th instars or melanized pupae produced from colonies that were initially infected in the laboratory, resulted in infections of 83% of the colonies, thus perpetuating the infection in other colonies. Infection was detected in 2 of 6 colonies after introducing adult worker caste ants that had died with V. invictae. The average number of adults and the volume of immature ants per colony were significantly lower in the infected than in the control colonies. Infected colonies had 86% fewer adults per colony and 82% less immature ants than the controls. A portion of the 16S rRNA gene of the V. invictae identified from these studies was amplified, cloned, and sequenced; the 1251 nucleotide amplicon was 100% identical to the 16S rRNA gene sequence recorded previously in the GenBank database, thus verifying the species as V. invictae. This is the first report of the artificial transmission of this pathogen to uninfected ant colonies, and demonstration of its ability to hinder growth in individual colonies.     

Impact of Thelohania solenopsae (Microsporidia: Thelohaniidae) on polygyne colonies of red imported fire ants (Hymenoptera: Formicidae)

Three studies were conducted to assess the effects of the entomopathogen Thelohania solenopsae on polygynous, red imported fire ant, Solenopsis invicta Buren, colonies. A total of 57 of 122 queens (46.7%) from nine, field-collected, polygyne, S. invicta colonies, was infected with T. solenopsae. Infection rate of queens for each colony ranged from 25 to 75%. Laboratory colonies of polygyne S. invicta, with three to 12 queens, were inoculated and infected with T. solenopsae. Brood levels in all infected colonies declined to 0 after 26-52 wk. Brood did not reappear in any of the colonies after 3-11 wk, even though in two of the eight infected colonies, five fertile queens that were uninfected were recovered. Thus, polygyne, S. invicta colonies infected with T solenopsae, which were confined and isolated under laboratory conditions, did not recover. Field plots that contained polygynous S. invicta colonies, which were infected with T solenopsae, were monitored over a 2-yr period. Infection rates increased during the study and reached a maximum of 9.3%. Fire ant nest density and colony sizes fluctuated over time, with maximum reduction of 63% per plot. In general, fire ant reductions were attributed to smaller colony sizes. T. solenopsae infections in polygynous S. invicta can result in a slow colony decline and death. Under field conditions, the prolonged colony death may mask the impact of T. solenopsae by allowing for concurrent reinfestations.     

Prevalence and impact of the microsporidium Thelohania solenopsae (Microsporidia) on wild populations of red imported fire ants, Solenopsis invicta, in Louisiana

We surveyed 165 sites to determine the ecological factors that might influence the distribution and prevalence of Thelohania solenopsae, and its effect on the demography of the red imported fire ant (Solenopsis invicta) in Louisiana. The microsporidium was found in 9.9% of colonies and at 16% of sites. Its distribution was clumped within the state with the majority of infected colonies and sites occurring in two infection patches. The proportion of polygyne colonies was a strong (positive) predictor of the proportion of infected colonies at a site. Infected monogyne colonies, however, still accounted for nearly 20% of infected colonies, a much higher proportion than anticipated. Several other factors, including the numbers of colonies at a site, precipitation, proximity to commercial waterways and ports, and type of habitat were also retained in the multiple logistic regression model describing T. solenopsae prevalence. The microsporidium appears to adversely affect the occurrence of brood, and possibly the size of S. invicta colonies and the mass of workers. It, however, was not included in the multiple regression model of the number of colonies or the density of ants at a site. Although our findings do not imply causation, they have identified several variables that might influence the epizootiology of T. solenopsae. Future work should concentrate on experimentally manipulating these variables to confirm these relationships.     

Susceptibility and behavioral response of red imported fire ant (Hymenoptera: Formicidae) to selected entomogenous nematodes (Rhabditida: Steinernematidae & Heterorhabditidae).

Pathogenicity of infective juveniles of selected Steinernema spp. and Heterorhabditis spp. toward developing and reproductive stages of the red imported fire ant, Solenopsis invicta Buren, was tested under laboratory conditions. At 10(3)-10(5) infective juveniles per Petri dish, mortality of reproductive larvae, pupae, and alates ranged from 28 to 100% at higher doses after 96 h at 23-25 degrees C. Steinernema carpocapsae All was the most consistent species tested; this nematode caused mortality of fire ant larvae, pupae, and alates of 82-94, 64-96, and 38-99%, respectively. Although not susceptible to nematode infection, worker ants vigorously preened nematodes from brood, alates, and themselves. In a field study, S. carpocapsae (5 x 10(6) and 2 x 10(6) drench, 2 x 10(6) infective juvenile infection) was applied to active fire ant mounds in 3.8-liter suspensions. Hydramethylnon (75 ml), a water drench, a water injection, and untreated fire ant mounds were marked and treated. Overall activity in mounds treated with nematodes of hydramethylnon ranged from 40 to 48%. Satellite mound activity accounted for 32-44% of overall activity in mounds treated with nematodes 2 wk after treatment. However, 6 wk after treatment, activity in mounds treated with hydramethylnon was 44%; activity of mounds treated with nematodes ranged from 52 to 80%. Satellite mound activity accounted for 0-24% of overall activity. Whereas a soil drench of S. carpocapsae showed potential as a control method for the red imported fire ant, colony relocation after nematode treatment could limit overall efficacy unless application techniques are developed to overcome or take advantage of the movement.     

蚁丘被破坏程度对红火蚁蚁群迁移的影响

本文调查研究了蚁丘被破坏的程度对红火蚁Solenopsis invicta Buren蚁群迁移的影响。结果表明,蚁巢地表部分被破坏可导致红火蚁蚁群发生分巢、搬巢等现象,蚁群的移巢比率随着蚁丘被破坏程度的增大而增大。当蚁丘体积被破坏75%时分巢比率最高,为14.44%;被破坏100%时搬巢比率、移巢比率均最高,分别为16.27%、27.00%。红火蚁蚁丘被破坏程度(D)与蚁群迁移比率(M)之间关系模型为M=4.3443+25.474D-3.4857D2。蚁丘受到破坏后,蚁群迁移距离平均为4.3m,迁移方向是随机的。     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Mechanisms of interspecific competition among an invasive and two native fire ants

The mechanisms of interspecific competition among an invasive and two native Solenopsis fire ant forms were investigated in a series of laboratory experiments. In separate trials each with a different food resource, the native S. geminata × xyloni retrieved the greatest amount of a protein- and lipid-rich artificial food resource and a high protein natural food resource, and the native S. geminata retrieved the greatest amount of a high carbohydrate food resource. In trials investigating aspects of interference competition at the colony level, the invasive S. invicta proved to be initially more aggressive than S. geminata , but less aggressive than S. geminata × xyloni . Solenopsis invicta eventually controlled more of the foraging arenas against both native forms when colonies were equivalent by worker biomass, but not when colonies were equivalent by worker number. When paired with S. invicta , S. geminata suffered a significantly greater proportional reduction in both workers and entire colonies when colonies were initially standardized by worker biomass, but not when colonies were standardized by worker number. When paired with S. invicta , a significantly greater proportional reduction of workers occurred in S. geminata × xyloni , regardless of how colonies were standardized. In pairwise trials at the individual level, majors always exhibited significantly less mortality than minors, regardless of the Solenopsis form. The majors of both native forms suffered significantly less mortality than those of S. invicta . Superiority in colony-level interference ability appears to be an important mechanism allowing S. invicta to displace native Solenopsis forms. The ability of S. invicta to reach high population densities, because of intrinsic biological characteristics or an escape from natural enemies, plays an important contributory role. Similar mechanisms may underlie the success of other invasive ant species.     

Use of ribosomal DNA sequence data to characterize and detect a neogregarine pathogen of Solenopsis invicta (Hymenoptera: Formicidae)

A neogregarine parasite of the red imported fire ant, Solenopsis invicta, was discovered recently in Florida and tentatively placed in the Mattesia genus based on morphological characterization. S. invicta infected with this Mattesia species exhibited a characteristic yellowing of the cuticle which was designated Mattesia "yellow-head disease" (YHD). The 18S rRNA gene sequence from Mattesia YHD was elucidated and compared with the neogregarine pathogens, Mattesia geminata and Ophriocystis elektroscirrha. The sequence data support the previous conclusion that Mattesia YHD is a new species that infects S. invicta. Furthermore, high sequence identity between Mattesia YHD, M. geminata (95.7%), and O. elektroscirrha (86.2%) correctly place the YHD organism in the Mattesia genus and Neogregarinorida order. Oligonucleotide primer pairs were designed to unique areas of the 18S rRNA genes of Mattesia YHD and S. invicta. Multiplex PCR resulted in sensitive and specific detection of Mattesia YHD infection of S. invicta.     

Microsatellite genotyping of red imported fire ant (Hymenoptera: Formicidae) colonies reveals that most colonies persist in plowed pastures

Our study focused on colony dynamics of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), in relation to the standard practice of planting rye grass (i.e., plowing) in the fall in Louisiana. Microsatellite molecular markers were used to determine genotypes of individuals from red imported fire ant colonies. These markers allowed us to monitor treatment effect by detecting changes in number and location of colonies in response to disking of pasture plots. Previous research on mound disturbance as a form of cultural control in pastures has produced mixed results. We found that the majority of colonies persisted on plots after plowing. Mound density and mound area, 5 mo after plowing, were not significantly different among treatments. In contrast, April measurements of mound volume were significantly smaller on plowed plots compared with control plots. A closer look at the rebuilding of mounds on plowed plots, during the 5 mo, showed that mound heights stayed below pretreatment measurements and they were significantly smaller than those of undisturbed mounds. Whether plowing has potential for use as a cultural control technique in reducing the impact of red imported fire ant mounds on agricultural practices in pastures remains to be seen. Conceivably, the best application of this technique will be in combination with other control measures in an integrated pest management approach to control red imported fire ants in pastures.     

Aggressive interactions between Solenopsis invicta and Linepithema humile (Hymenoptera: Formicidae) under laboratory conditions

The Argentine ant, Linepithema humile (Mayr), and the red imported fire ant, Solenopsis invicta Buren, are natural agonists in their country of origin. Since the first report of L. humile in California in 1907 its range expanded statewide, displacing native ant species wherever it spread. Since the discovery of established populations of S. invicta in southern California in 1998, it has been restricted to discrete areas of southern California. However, as these discrete populations expand, they are encountering large populations of L. humile, which are effective competitors for available resources and are particularly aggressive in their encounters with other ant species such as S. invicta. Most Dolichoderine ants such as L. humile do not prefer to forage on baits made with defatted corn grit and soybean oil typically used in red imported fire ant control programs. Applications of these baits in areas where distributions of these species overlap might selectively affect populations of S. invicta and give L. humile a competitive advantage. Three laboratory experiments were conducted to determine the competitive outcomes between S. invicta pitted against L. humile: 1) agonistic behavior of workers in small arenas, 2) colony interactions with different population ratios, and 3) the effects of pyriproxyfen on the competitiveness of S. invicta against L. humile. Populations of S. invicta consisting of major workers killed more L. humile than did minors or a mixture of majors and minors. When paired against L. humile colonies consisting of 1,100 workers, colonies consisting of 38 S. invicta workers were easily defeated by L. humile. Colonies consisting of 450 S. invicta workers plugged their nest entrances, but they were ultimately defeated by L. humile after 13 d. The S. invicta colonies consisting of 1,100 workers took control of the bridge connecting the colonies, invaded the L. humile colony, killed the Argentine ant queens, and removed their brood. Pyriproxyfen-treated fire ant workers took significantly longer to chase the Argentine ants from a connecting bridge than did untreated fire ants. Thus, fire ant baits may have long-term effects on intercolonial aggression between S. invicta and L. humile, especially when Argentine ant populations are high in the summer.     

Genome complexity and organization in the red imported fire ant Solenopsis invicta Buren

The red imported fire ant Solenopsis invicta is the most destructive invading arthropod in the southern United States, yet little is known about its genome complexity and organization. Here we report the size, organization and GC content of S. invicta genome. DNA reassociation kinetics using S1 nuclease assay and a modified second-order kinetics model indicated that the S. invicta genome is approximately 0.62 picograms or 5.91 x 10(8) base pairs, composed of 36% unique, 41% moderately repetitive and 23% highly repetitive/foldback sequences. Comparison of the reassociation kinetics of short and long DNA fragments revealed that the sequence arrangement follows a pattern of short period interspersion, as in most organisms with relatively large genomes. Melting-temperature analysis showed that the GC content of the fire ant genomic DNA is 34.8%, similar to that of most eukaryotic organisms. The results reveal that the fire ant genome is much larger and more complex than those of a number of hymenopteran insects studied to date. Our study provides a foundation for further analysis and genetic manipulation of the S. invicta genome.