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.     

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.     

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 Threonine¹²⁶⁶-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine¹²⁶⁶ group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine¹²⁶⁶/Valine¹²⁸⁵ and Serine¹²⁶⁶/Isoleucine¹²⁸⁵ combinations are predominant phenotypes.     

Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta

Loss of natural enemies during colonization is a prominent hypothesis explaining enhanced performance of invasive species in introduced areas. Numerous studies have tested this enemy release hypothesis in a wide range of taxa but few studies have focused on invasive ants. We conducted extensive surveys for the presence of six microbes in recently established populations (California, Australia, New Zealand, Taiwan, and China) of the invasive fire ant Solenopsis invicta. These microbes include Wolbachia, two microsporidia (Kneallhazia solenopsae and Vairimorpha invictae) and three RNA viruses (SINV-1, -2 and -3), all of which previously have been reported in native South American populations of S. invicta. These surveys showed that the total number of enemy species is lower in the recently invaded areas compared with both South American and US populations. Only two microbes were found in any of these recently invaded areas: SINV-1 was detected in all surveyed populations except Australia and New Zealand, and SINV-2 was detected in California and Taiwan only. These results support the general prediction that invasive species lose many of their natural enemies during invasion. Further, the conspicuous absence of some of these microbes in these areas may result from strong selection against founders due to fitness costs associated with harboring detrimental infections rather than the alternative hypothesis that they simply were absent among the original founders. While the successful invasion of S. invicta in these recently invaded areas may be explained partly by the absence of natural enemies, other factors likely have been important as well.     

New feasible quarantine disinfestation using ethyl formate for termites and ants on imported lumber

The interception of exotic ants and termites from stone and lumber imported into Korea is on the rise, including Solenopsis invicta. Currently, a relatively long exposure (24 h) of methyl bromide (MB) fumigation is the only practical disinfestation option for exotic ants and termites on imported stone and lumber. In this study, ethyl formate (EF) wase evaluated as a potential MB alternative to disinfest ants and termites in imported lumber, with a focus on S. invicta. As S. invicta is not established in Korea, we first determined whether Reticulitermes speratus, established in Korea, could be considered as an EF surrogate of S. invicta. From 1 h EF fumigation trials, workers of S. invicta were more sensitive to EF than those of R. speratus based on lethal concentration × time causing 99% mortality (LCt_99%), supporting that R. speratus can be used as an EF surrogate of S. invicta. When R. speratus workers were further fumigated with EF for 4 h at four different temperatures, LCt_99% values for R. speratus workers were 108.00, 63.31, 77.24, and 67.24 g h/m³ at 2, 5, 13, and 23 °C, respectively. From scale-up (0.65 m³) and commercial scale (76.4 m³) trials containing two infestation-prone lumber species, lauan (Shorea spp.) and acacia (Acacia spp.), 140 g/m³ EF at >5 °C for 4 h resulted in the complete control of R. speratus workers, suggesting that 140 g/m³ 4 h-EF-fumigation may be applicable as a potential MB-alternative treatment for disinfestation of invasive S. invicta on imported lumber.     

Interspecific competition between the red imported fire ant,Solenopsis invicta Buren and ghost ant,Tapinoma melanocephalum Fabricius for honeydew resources produced by an invasive mealybug,Phenacoccus solenopsis Tinsiley

In natural as in agricultural ecosystems, interactions between ants and honeydew-producing hemipterans are commonly observed. Mutualisms between invasive ants and hemipterans have been extensively studied in recent years. However, native ant species can equally exploit the honeydew excreted by hemipterans, and establish close relationships with them. Up till present, little is known about the competition between exotic ants (such as Solenopsis invicta) and its co-occurring species (e.g., Tapinoma melanocephalum) for this food resource. In this study, we compared the competitive ability of the invasive ant S. invicta and its co-occurring species T. melanocephalum in the laboratory. We also determined whether the two ant species could coexist and share honeydew resource. Our results indicate that the foraging activity of T. melanocephalum was restrained by S. invicta. Mortality of S. invicta and T. melanocephalum was significantly higher in T. melanocephalum colony case than that in other cases. The invasive ability between the two ant species was significantly different. These results suggest that S. invicta suppresses exploitation of honeydew-producing hemipterans by native ants and occupies most of honeydew resource. S. invicta could not completely drive T. melanocephalum out of honeydew competition, with small numbers of T. melanocephalum workers coexisting and sharing the honeydew with S. invicta. This finding permits a better understanding of the invasion success of S. invicta, and its ability to occupy new habitats.     

The Effect of Diet and Opponent Size on Aggressive Interactions Involving Caribbean Crazy Ants (Nylanderia fulva)

Biotic interactions are often important in the establishment and spread of invasive species. In particular, competition between introduced and native species can strongly influence the distribution and spread of exotic species and in some cases competition among introduced species can be important. The Caribbean crazy ant, Nylanderia fulva, was recently introduced to the Gulf Coast of Texas, and appears to be spreading inland. It has been hypothesized that competition with the red imported fire ant, Solenopsis invicta, may be an important factor in the spread of crazy ants. We investigated the potential of interspecific competition among these two introduced ants by measuring interspecific aggression between Caribbean crazy ant workers and workers of Solenopsis invicta. Specifically, we examined the effect of body size and diet on individual-level aggressive interactions among crazy ant workers and fire ants. We found that differences in diet did not alter interactions between crazy ant workers from different nests, but carbohydrate level did play an important role in antagonistic interactions with fire ants: crazy ants on low sugar diets were more aggressive and less likely to be killed in aggressive encounters with fire ants. We found that large fire ants engaged in fewer fights with crazy ants than small fire ants, but fire ant size affected neither fire ant nor crazy ant mortality. Overall, crazy ants experienced higher mortality than fire ants after aggressive encounters. Our findings suggest that fire ant workers might outcompete crazy ant workers on an individual level, providing some biotic resistance to crazy ant range expansion. However, this resistance may be overcome by crazy ants that have a restricted sugar intake, which may occur when crazy ants are excluded from resources by fire ants.     

Solenopsis invicta virus-1 tissue tropism and intra-colony infection rate in the red imported fire ant: a quantitative PCR-based study

Quantitative real-time PCR was employed to measure the Solenopsis invicta virus 1 (SINV-1) load in tissues, individuals, and among colonies of the red imported fire ant, S. invicta Buren. Among tissues examined from SINV-1-infected adults and larvae, the alimentary canal (specifically the midgut) consistently had the highest number of SINV-1 genome copies (91.1 and 99.9%, respectively). Negative staining of a supernatant of the gut homogenate demonstrated the presence of spherical virus particles with a diameter of 30-35 nm, consistent with SINV-1. The number of SINV-1 genome copies in infected larvae and workers from the same queenright colonies were similar to each other. In other words, the infection rate was consistent among both developmental stages. No significant differences were observed in SINV-1 genome copy number among infected colonies sampled during the winter and summer. Although the SINV-1 infection rate of summer-collected mounds was previously shown to be six-times higher than winter-collected mounds, the intra-colony infection rate appears to be unaffected by season. Perhaps less inter-mound interaction during the winter months among S. invicta restricts spread of the virus. A positive correlation between intra-colony infection rate and mean SINV-1 genome copy number per ant was also observed. Based on these results, it is likely that SINV-1 replicates in gut epithelia of S. invicta and virus is shed into the gut lumen where it may be transmitted to nestmates by trophallaxis.     

The evolution of genome size in ants

Background  

Despite the economic and ecological importance of ants, genomic tools for this family (Formicidae) remain woefully scarce. Knowledge of genome size, for example, is a useful and necessary prerequisite for the development of many genomic resources, yet it has been reported for only one ant species (Solenopsis invicta), and the two published estimates for this species differ by 146.7 Mb (0.15 pg).     

Imported crazy ant displaces imported fire ant, reduces and homogenizes grassland ant and arthropod assemblages

A recently introduced, ecologically dominant, exotic ant species, Nylanderia fulva, is invading the Southeastern United States and Texas. We evaluate how this invader impacts diversity and abundance of co-occurring ants and other arthropods in two grasslands. N. fulva rapidly attains densities up to 2 orders of magnitude greater than the combined abundance of all other ants. Overall ant biomass increases in invaded habitat, indicating that N. fulva exploits resources not fully utilized by the local ant assemblage. At high density, as N. fulva spreads, it eliminates the current ecologically dominant invasive ant, red imported fire ants (Solenopsis invicta). Compared to imported fire ant dominated habitat, N. fulva invasion zones have lower non-ant arthropod species richness and abundance with impacts differing by trophic category. Further, N. fulva reduces abundance and species richness of the remainder of the ant assemblage and does so in a non-random manner: impacting species with small sized workers much less than species with larger workers. In these and other ant assemblages with a large exotic component, the exotics tend to be small bodied species. As a result, N. fulva almost completely eliminates regionally distributed species, but leaves globally distributed species largely unaffected, thereby systematically favoring introduced over native diversity. S. invicta impacts wildlife and arthropod assemblage structure and is nearly ubiquitous in non-forested habitats of the Southeastern United States and Texas. Its displacement by N. fulva has critical implications for the natural systems of this region.     

Vitellogenin Underwent Subfunctionalization to Acquire Caste and Behavioral Specific Expression in the Harvester Ant Pogonomyrmex barbatus

The reproductive ground plan hypothesis (RGPH) proposes that the physiological pathways regulating reproduction were co-opted to regulate worker division of labor. Support for this hypothesis in honeybees is provided by studies demonstrating that the reproductive potential of workers, assessed by the levels of vitellogenin (Vg), is linked to task performance. Interestingly, contrary to honeybees that have a single Vg ortholog and potentially fertile nurses, the genome of the harvester ant Pogonomyrmex barbatus harbors two Vg genes (Pb_Vg1 and Pb_Vg2) and nurses produce infertile trophic eggs. P. barbatus, thus, provides a unique model to investigate whether Vg duplication in ants was followed by subfunctionalization to acquire reproductive and non-reproductive functions and whether Vg reproductive function was co-opted to regulate behavior in sterile workers. To investigate these questions, we compared the expression patterns of P. barbatus Vg genes and analyzed the phylogenetic relationships and molecular evolution of Vg genes in ants. qRT-PCRs revealed that Pb_Vg1 is more highly expressed in queens compared to workers and in nurses compared to foragers. By contrast, the level of expression of Pb_Vg2 was higher in foragers than in nurses and queens. Phylogenetic analyses show that a first duplication of the ancestral Vg gene occurred after the divergence between the poneroid and formicoid clades and subsequent duplications occurred in the lineages leading to Solenopsis invicta, Linepithema humile and Acromyrmex echinatior. The initial duplication resulted in two Vg gene subfamilies preferentially expressed in queens and nurses (subfamily A) or in foraging workers (subfamily B). Finally, molecular evolution analyses show that the subfamily A experienced positive selection, while the subfamily B showed overall relaxation of purifying selection. Our results suggest that in P. barbatus the Vg gene underwent subfunctionalization after duplication to acquire caste- and behavior- specific expression associated with reproductive and non-reproductive functions, supporting the validity of the RGPH in ants.     

Light and electron microscope study of Thelohania solenopsae n. sp. (Microsporida: Protozoa) in the red imported fire ant,Solenopsis invicta

A new species of Microsporida, Thelohania solenopsae, is described from the red imported fire ant, Solenopsis invicta. The Thelohania infections are localized in the fat body of workers. Meronts causing infections of progeny are found in the ovaries of queens. Spores occur only in adult ants and only vegetative stages are present in larvae and pupae. Both uninucleate octospores (eight spores within a pansporoblast membrane) and binucleate free spores (spores developing in isolation) are formed.     

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.     

Cover Caption

The red imported fire ant, Solenopsis invicta Buren, is an important medical, agricultural, and ecological pest with a global distribution. The main food source of S. invicta is liquid carbohydrates (e.g., plant exudates and honeydew excreted by Hemipterans). This species evolved many strategies to forage droplets of liquid food. The photo shows that S. invicta workers surrounded and fed along the edge of the sucrose water droplet in the field. The diagrammatic sketch (at the bottom of the figure) shows how ants stacked to feed the droplet in the laboratory. After the edge of the droplet was ‘saturated’ by surrounding ants, the stacking ants stepped on other individuals and climbed onto the droplet (see pages 499–507). Blue, red, and green arrows indicate surrounding ants, stacking ants, and incoming ants, respectively. Photo by Wen‐Quan Qin and Qin‐Xi Xie.     

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.     

Red imported fire ants (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>) and seasonality influence community refuge use

Refuges are fundamental to animal ecology as refuge availability affects many levels of biological organization—from the behavior and physiology of individuals to the interspecific dynamics of a community. Although frequently studied in the context of predator–prey interactions, refuges may also mediate interspecific competition between native and invasive taxa given the role of refuges as a valuable resource. Because interspecific interactions (e.g., competition and predation) can be modulated by temporal and biotic (e.g., trophic level) factors, we used a manipulative approach to investigate community-wide refuge-use patterns in the context of two important ecological factors: invasive species and seasonality. We surveyed refuge (artificial cover object) use of ants and vertebrates in a forest community for 2 years, and we systematically suppressed an established invasive species (red imported fire ant, Solenopsis invicta) to examine its impact on community refuge use. Native Camponotus ants appeared to co-exist and share refuges with S. invicta, but we found evidence for a negative effect of S. invicta on vertebrate refuge use that was also influenced by season. Vertebrates were more abundant under refuges undergoing suppression of S. invicta, and they were less abundant under refuges during the fall (the season characterized by the highest occupancy of refuges by S. invicta). Thus, researchers must continue to examine the entire community and to incorporate the effects of season when assessing the impact of invasive species (e.g., at our site, a survey conducted only in the summer or only on native ants would have indicated a negligible effect of S. invicta on community refuge use).     

Individual and Cooperative Food Transport of the Red Imported Fire Ant (Hymenoptera: Formicidae): Laboratory Observations

The red imported fire ant, Solenopsis invicta Buren, is a significant urban and agricultural pest with a global distribution. However, the food transport patterns of S. invicta are poorly understood. In the present study, food of different sizes were provided to S. invicta colonies and transport for a short distance was observed under laboratory conditions. Individual transport of small food (1 × 1 mm deli meat) and cooperative transport of food of larger sizes (2 × 2 mm, 4 × 4 mm, and 8 × 8 mm deli meat) was observed. The majority of transporters were small workers. During cooperative transport, ants were either around or on the food, pulling/pushing the food item in multiple directions, leading to a slow process with frequent deadlocks. Individual transport required significantly shorter time in the preparation and transportation stages, but food transport was more likely to deviate from the nest. This observation suggests that the cooperative transport of S. invicta is closer to uncoordinated.