New Family,Genus, and Species of Microsporida (Protozoa: Microsporida) from the Tropical Fire Ant,Solenopsis geminata (Fabricius) (Insecta: Formicidae)*

SYNOPSIS. A new species of Microsporida, Burenella dimorpha sp. n., representing a new family, Burenellidae fam. n. and genus, is described on the basis of light- and electron-microscope observations. The family is characterized by 2 sequences of sporogony, each sequence having morphologically different sporonts and spores. The parasite infects the tropical fire ant, Solenopsis geminata (Fabricius), producing distinct pathologic manifestations (clearing of the cuticle and eye malformation) and death in the pupal stage of development. Transmission of the infection per os to healthy S. geminata, to the Southern fire ant, Solenopsis xyloni McCook, and to the red and black imported fire ants, Solenopsis invicta Buren and Solenopsis richteri Forel, is reported.     

Pseudacteon decapitating flies (Diptera: Phoridae): Are they potential vectors of the fire ant pathogens Kneallhazia (=Thelohania) solenopsae (Microsporidia: Thelohaniidae) and Vairimorpha invictae (Microsporidia: Burenellidae)?

Fire ant decapitating flies in the genus Pseudacteon were tested for their potential as hosts or vectors of two microsporidian pathogens of the red imported fire ant, Solenopsis invicta. Decapitating flies that attacked or were reared from S. invicta workers infected by Kneallhazia (=Thelohania) solenopsae or Vairimorpha invictae were tested for either pathogen by polymerase chain reaction (PCR) tests or visual examination for spores using phase microscopy. Three species of fire ant decapitating flies acquired the pathogen, Kneallhazia solenopsae. K. solenopsae was detected in 58% of pooled samples of Pseudacteon obtusus flies and 44% of pooled samples of Pseudacteon cultellatus that developed in K. solenopsae-infected fire ant workers. K. solenopsae was also found in 17% of pooled samples of field-collected Pseudacteon curvatus. In contrast, the microsporidium V. invictae was not detected in P. obtusus reared from V. invictae-infected S. invicta workers. Neither K. solenopsae nor V. invictae were detected in any of the hovering or ovipositing flies in the laboratory exposures, indicating no mechanical acquisition of the microsporidia occurred during oviposition activity. Greater than 92% of the P. obtusus that developed in K. solenopsae-infected ants survived and emerged as adults, thus indicating no detrimental effects of the microsporidium on pupal development and emergence. These results indicate that Pseudacteon decapitating flies may be able to vector K. solenopsae but not V. invictae among fire ants. Further tests are planned to determine if flies containing K. solenopsae are capable of transferring this pathogen either during oviposition or by being consumed by fire ant larvae.     

COMPARATIVE BIOCHEMICAL GENETICS OF THREE FIRE ANT SPECIES IN NORTH AMERICA,WITH SPECIAL REFERENCE TO THE TWO SOCIAL FORMS OF SOLENOPSIS INVICTA (HYMENOPTERA: FORMICIDAE)

An electrophoretic study of the genetics of three fire ant species in North America was undertaken with the primary objective of further clarifying the genetic relationship between two social forms of Solenopsis invicta. Such social forms are common in many groups of ants and may, in some cases, represent significant intermediate stages in the speciation process. The monogyne and polygyne forms of S. invicta, while differing in a number of important biological traits, are genetically indistinguishable, in contrast to the substantial genetic differentiation observed between S. invicta and a second, closely related, introduced species, S. richteri. The native fire ant, S. geminata, is genetically the most distinct of the three species studied, in accord with its taxonomic placement in a different species complex. Hypotheses concerning the derivation of the polygyne form of S. invicta from the monogyne form which invoke their long-term reproductive isolation in South America and separate introductions to North America appear unfounded. Although S. invicta and S. richteri are known to hybridize in North America, our study provided no evidence of gene introgression between S. invicta and the native species, S. geminata, in areas where our samples were collected. Analyses of population structure in S. invicta failed to reveal significant differentiation of populations or local inbreeding. Levels of genetic diversity in the three species studied, although not significantly different, were in the order predicted from knowledge of the population biology and recent history of the taxa, with S. richteri exhibiting the least diversity, S. geminata the greatest, and S. invicta having an intermediate level.     

Kneallhazia carolinensae sp. nov., a microsporidian pathogen of the thief ant, Solenopsis carolinensis

A new species of microsporidia is described from adults of the thief ant, Solenopsis carolinensis, collected in Florida, USA. Morphological and genetic characterization of this new species showed that it is most closely related to the genus Kneallhazia and is therefore formally designated, Kneallhazia carolinensae sp. nov. Masses of ovoid, binucleate spores were localized to fat body of adult workers and measured 6.2 ± 0.1 × 3.1 ± 0.1 μm (fresh) and 6.0 ± 0.1 × 3.4 ± 0.1 μm (fixed). These spores were in direct contact with the cell cytoplasm and contained an isofilar polar filament with 12-15 coils. Blastn analysis revealed that the K. carolinensae 16S rDNA sequence exhibited 91% identity with the 16S rDNA gene of K. solenopsae. The morphological and sequence data support the conclusion that K. carolinensae is a novel microsporidian species distinct from K. solenopsae.     

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.     

Reproduction by virgin queen fire ants in queenless colonies: Comparative study of three taxa (Solenopsis richteri,hybridS. invicta/richteri,S. geminata) (Hymenoptera: Formicidae)

Summary In the fire ant,Solenopsis invicta, some winged virgin queens are known to shed their wings (dealate) upon removal of the mated mother queen. These virgin queens then develop their ovaries and begin to lay eggs, thereby foregoing the option of leaving on mating flights and attempting to found their own colonies. Such a response of virgin queens to queenlessness has not been reported for other ants. In order to determine if virgin queens of some other fire ants (subgenusSolenopsis) would respond in the same way, experiments were conducted onS. richteri, hybridS. invicta/richteri andS. geminata, a member of a species complex different from that of the other taxa. Just as inS. invicta, virgin queens ofS. richteri and the hybrid dealated and began to lay eggs within days of the removal of the queen. In addition, workers executed many of the reproductively active virgin queens, a phenomenon also found inS. invicta. In contrast, virgin queens ofS. geminata did not dealate or quickly begin to lay eggs upon separation from the queen. Reasons for the variability in the response of virgin queens of the different species may be 1) higher probability of reproductive success for unmated dealated queens compared to normal claustral founding inS. invicta andS. richteri linked to relatively frequent loss of the mother queen; or 2) phylogenetic constraint.     

The urban fire ant paradox: native fire ants persist in an urban refuge while invasive fire ants dominate natural habitats

In contrast to the widespread extirpation of native fire ants (Solenopsis geminata) across southern US following the invasion by imported red fire ants (S. invicta), some residential areas of Austin form unexpected refuges for native fire ants. Ironically, these urban environments provide refuges for the native fire ants while adjacent natural habitats have been overrun by invasive fire ants. Resistance to invasive fire ants in these urban areas occurs mainly in older residential properties constructed prior to the S. invicta invasion, while more recent construction has allowed establishment by S. invicta. The invasive ability of S. invicta is often attributed to escape from parasitoids and efficient dispersal of polygyne multiple queen colonies. Here we also show the importance of landscape parameters in the invasion process, where low levels of disturbance and continuous plant cover in older residential areas form possible barriers to colonization. Dense leaf cover (high NDVI) was also found to be associated with native ant refuges. Long term residential land ownership may have resulted in lower recent disturbance levels and increased plant cover that support refuges of native fire ants.     

Host preference in Pseudacteon phorid flies: response of P. tricuspis and P. curvatus to black, red and hybrid imported Solenopsis fire ants in multiple choice bioassays

Host preferences in both sexes of Pseudacteon tricuspis Borgmeier (Jaguariuna biotype) and Pseudacteon curvatus Borgmeier (Formosa biotype) and their relative attraction to the imported fire ants (IFA), Solenopsis invicta Buren (red IFA), Solenopsis richteri Forel (black IFA) and S. invicta × S. richteri hybrids (hybrid IFA) were investigated in two separate experiments utilizing multiple choice flight bioassays. The results of both experiments clearly showed that both sexes of the Jaguariuna biotype of P. tricuspis could distinguish among the three IFA species and demonstrated greater preference for hybrid IFA and red IFA. This conclusion is supported by a variety of data collected on the number of fly visits, attack rate, and hovering duration (Experiment 1), and on the number of trapped flies (Experiment 2), which showed that black IFA is the least preferred of the three species. Similar results were recorded for the Formosan biotype of P. curvatus, although the data were not as strongly conclusive. Females of this biotype spent a significantly greater amount of time in hovering mode over red IFA and hybrid IFA compared to black IFA, but the other data were not significant. The red IFA is the natural host of both phorid fly biotypes and our results suggest that both biotypes may have evolved a specialized relationship with red IFA including an ability to discriminate it from related fire ants. These results are discussed in relation to the possible role of fire ant chemicals in mediating host preferences in phorid flies, contributions of male phorid flies to fire ant biocontrol, and the practical implications of the key findings.     

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.     

Fire ant trail pheromones: Analysis of species specificity after gas chromatographic fractionation

The specificity of the trail pheromones of four Solenopsis species was determined using natural trails. Dufour's gland extracts, and purified fractions from Dufour's gland extracts collected after gas-chromatographic separation. S. richteri and S. invicta possess species-specific major trail pheromones, while S. geminata and S. xyloni appear to have a common trail pheromone. Preliminary chemical characterization of the main trail pheromone of S. richteri indicates a M.W. of 218 and empirical formula of C₁₆H₂₆. The trail pheromone system of S. richteri consists of a blend of compounds and this phenomenon may also occur in the other species. The lowest concentration of their trail pheromone that workers of S. richteri could detect was about 10 fg per cm. The significance of blends of pheromones being utilized to generate chemical trails is discussed.     

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.     

A South American fire ant,Solenopsis nr. saevissima,in Guadeloupe,French West Indies

Several species of fire ants, well known for their painful stings, have spread from their native ranges and have become invasive pests, notably Solenopsis geminata, Solenopsis invicta, and Solenopsis richteri. Here, I report the first known exotic spread of another fire ant, Solenopsis nr. saevissima, a South American species now established on the French West Indian island of Guadeloupe. In 2008, I first found this shiny yellow fire ant at Plage de Viard beach on Guadeloupe. Genetic analyses indicated that this species was close to the yellow–brown variant of S. saevissima known only from Western Amazonia, but not an exact match. I returned to Guadeloupe in 2011 and found S. nr. saevissima at Plage de Viard plus five additional sites along a >20 km stretch on the east coast of Basse-Terre, from the Jarry commercial shipping port in the north to Plage Roseau in the south. It seems most likely that S. nr. saevissima arrived in Guadeloupe from South America in a commercial shipment unloaded at the port in Jarry. It is unclear whether this species will develop into a major new problem. Solenopsis nr. saevissima may simply replace another pernicious fire ant, S. geminata, which apparently arrived in Guadeloupe from South America several 100 years ago.     

Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. They are successful invaders due to their preference for disturbed habitats, high reproductive rates, and the ability to feed on a wide variety of food items (omnivorous). Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of endosymbiotic bacteria possibly involved in nutrient distributions. Our study utilized 16S rDNA sequencing to describe the composition of the bacterial community in fourth-instar ant larvae in order to identify possible endosymbiotic bacteria present therein. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. Our results did not detect the presence of endosymbiotic bacteria in the guts of RIFA larvae among the colonies. In addition, minimal species overlap was found when bacterial inventories were compared among colonies. Thus, bacteria coadapted with red imported fire ant larvae were not detected. Identified bacteria were not closely affiliated with endosymbiotic bacteria common in other insect species. Bacteria communities appeared to be unique to each geographical location and were determined by the foods consumed by the ants.     

Fire ant alarm pheromone and venom alkaloids act in concert to attract parasitic phorid flies, Pseudacteon spp.

Pseudacteon tricuspis, Pseudacteon obtusus and Pseudacteon curvatus are three species of parasitic phorid flies (Diptera: Phoridae), which have been introduced as classical biological control agents of imported, Solenopsis fire ants (Hymenoptera: Formicidae) in the southern USA. Previous studies demonstrated the behavioral response of P. tricuspis to the venom alkaloids and alarm pheromone of the fire ant, S. invicta. In the present study, we compared the responses of P. tricuspis, P. obtusus and P. curvatus to Solenopsis invicta alarm pheromone, venom alkaloids, or a mixture of both chemicals in four-choice olfactometer bioassays. The main hypothesis tested was that the fire ant alarm pheromone and venom alkaloids act in concert to attract Pseudacteon phorid flies. Both sexes of all three Pseudacteon species were attracted to low doses of the fire ant alarm pheromone or venom alkaloids (i.e. 1 ant worker equivalent) alone. However, the flies were significantly more attracted to a mixture of both chemicals (i.e., 1:1 mixture of alarm pheromone + alkaloids) than to either chemical. The results suggest an additive rather than a synergistic effect of combining both chemicals. Comparing the fly species, P. tricuspis showed relatively greater attraction to cis alkaloids, whereas the alkaloid mixture (cis + trans) was preferred by P. obtusus and P. curvatus. In general, no key sexual differences were recorded, although females of P. tricuspis and P. obtusus showed slightly higher response than conspecific males to lower doses of the alarm pheromone. The ecological significance of these findings is discussed, and a host location model is proposed for parasitic phorid flies involving the use of fire ant alarm pheromone and venom alkaloids as long range and short range attractants, respectively.     

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.     

Potential Global Range Expansion of the Invasive Fire Ant, Solenopsis invicta

The red imported fire ant, Solenopsis invicta Buren, is an invasive pest that has become widespread in the southern United States and Caribbean after introduction from South America in the 1930s. This species, which has diverse detrimental impacts on recipient communities, was recently discovered in Australia and New Zealand and has the potential to colonize numerous other regions. We used a dynamic, ecophysiological model of colony growth to predict the potential global range expansion of this invasive species. Based on minimum and maximum daily temperatures, the model estimates colony alate production and predicts future geographic range limits. Because S. invicta populations are limited by arid conditions as well as cold temperatures, we superimposed precipitation data upon temperature-based predictions, to identify regions that do not receive enough rainfall to support this species across the landscape. Many areas around the globe, including large portions of Europe, Asia, Africa, Australia, and numerous island nations, are at risk for S. invicta infestation. Quarantine officials should be vigilant for any accidental introductions of this pest in susceptible regions. Costs of eradication increase dramatically as the area of infestation grows, and large infestations may be impossible to eradicate. Other South American Solenopsis fire ants (e.g., S. richteri Forel) may become invasive if the opportunity arises, and our predictions for S. invicta may approximate the potential range limits for these species as well.     

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.     

Post-Release Host-Specificity Testing of Pseudacteon tricuspis, a Phorid Parasitoid of Solenopsis Invicta Fire Ants

Inherent in any biological control program is the risk of nontarget effects. Pseudacteon tricuspisBorgmeier, a parasitoid phorid fly, has been introduced to the United States from South America as a potential biocontrol agent of the red imported fire ant, Solenopsis invictaBuren. We conducted tests of host specificity on introduced populations of P. tricuspis, which are attracted to alarm pheromones released by their hosts during events such as mound disturbances and interspecific interactions. We monitored disturbed mounds of S. invicta and its close congener, S. geminata(F.), during the expansion of P. tricuspis across north Florida and after populations had been established for ~3 years. We also tested host acceptance in established populations of P. tricuspis by offering trays containing S. invicta, S. geminata, and 14 additional ant species representing 12 different non-Solenopsis genera. Although P. tricuspiswas commonly observed to hover over and attempt to oviposit on S. invicta, we never observed any parasitization attempts on any other ant species. As predicted by laboratory tests, released populations of P. tricuspis appear to be highly host specific and pose no obvious threat to nontarget species.     

Zombie fire ant workers: behavior controlled by decapitating fly parasitoids

Laboratory observations were conducted on four separate red imported fire ant, Solenopsis invicta, colonies that contained workers parasitized by the decapitating fly, Pseudacteon tricuspis. Parasitized S. invicta workers remained inside the nest during parasitoid larval development and left the nest approximately 8 – 10 hours before decapitation by the parasitoid. When parasitized ants left the nest, they were highly mobile, were responsive to tactile stimuli, and showed minimal defensive behavior. Ants ultimately entered into a grass thatch layer, where they were decapitated and the fly maggots pupariated. This study reveals that parasitized ants exhibit behaviors that are consistent with host manipulation to benefit survival of the parasitoid. Received 9 November 2006; revised 26 January 2007; accepted 7 February.