Biology of Pseudacteon Decapitating Flies (Diptera: Phoridae) That Parasitize Ants of the Solenopsis saevissima Complex (Hymenoptera: Formicidae) in South America.

Pseudacteon flies (Diptera: Phoridae) parasitize individual ant workers, causing decapitation of the host during pupariation. Phorid flies that attack South American fire ants in the Solenopsis saevissima (Smith) complex are distributed across a wide range of habitats and climates associated with the geographical range of their hosts. Sympatric species sharing the same hosts often partition niche resources by season, active time of day, host size, and/or different host activities. They have the potential of being used for biological control of the imported fire ants in North America, Australia, and Asia.

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

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

Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina.

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.

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

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

Cuticular Hydrocarbon Profiles Differentiate Tropical Fire Ant Populations (Solenopsis geminata, Hymenoptera: Formicidae).

The cuticular hydrocarbons (CHCs) from hexane rinses of workers from two Florida populations (dark and red forms) of the tropical fire ant, Solenopsis geminata, were separated by silica gel chromatography and identified by GC/MS analysis. Both the dark form and the red form produce similar CHCs with carbon chain lengths ranging from 17 to 35. However, the relative percentages of these CHCs were consistently different between the two color forms. The largest CHC component in the dark form is tricosane, and (Z)-9-tricosene for the red form. There were several significant differences in percent composition. For example, the dark form was characterized by a low tricosene:tricosane ratio (ca. 0.25), whereas this ratio was > 2.5 for the red form. The ratio of tricosene:tricosane can be used as a diagnostic biomarker to delimit the dark and red forms. Cluster analysis showed that the CHCs patterns of dark form colonies are completely separated from the CHC pattern of red form colonies. Differences in social behaviors like nestmate recognition and polygyny between workers from this dark form and the red form await further investigation.

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

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

Solenopsis invicta virus 3: pathogenesis and stage specificity in red imported fire ants.

Solenopsis invicta colonies were exposed to purified preparations of Solenopsis invicta virus 3 (SINV-3) to investigate virus pathogenesis at the colony level. Time course experiments revealed an infection exhibiting specificity for the adult stage (workers). SINV-3 genome and a capsid protein were increasingly present in worker ants with time. Northern blot analysis revealed two bands in RNA preparations from worker ants infected with SINV-3 corresponding to the genomic and sub-genomic species. Conversely, larval RNA preparations from SINV-3-infected colonies showed a near-complete absence of SINV-3 genome or sub-genome. The data confirm that SINV-3 is the etiological agent causing mortality among S. invicta colonies in the laboratory. We propose that SINV-3 infection somehow alters worker ant behavior, which may prevent them from acquiring and/or distributing solid food to the larvae. Consequently, larval mortality and impaired queen health occur as a result of starvation or neglect by the worker caste.

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.

Fire ant decapitating fly cooperative release programs (1994-2008): two Pseudacteon species, P. tricuspis and P. curvatus, rapidly expand across imported fire ant populations in the southeastern United States.

Natural enemies of the imported fire ants, Solenopsis invicta Buren S. richteri Forel (Hymenoptera: Formicidae), and their hybrid, include a suite of more than 20 fire ant decapitating phorid flies from South America in the genus Pseudacteon. Over the past 12 years, many researchers and associates have cooperated in introducing several species as classical or self-sustaining biological control agents in the United States. As a result, two species of flies, Pseudacteon tricuspis Borgmeier and P. curvatus Borgmeier (Diptera: Phoridae), are well established across large areas of the southeastern United States. Whereas many researchers have published local and state information about the establishment and spread of these flies, here distribution data from both published and unpublished sources has been compiled for the entire United States with the goal of presenting confirmed and probable distributions as of the fall of 2008. Documented rates of expansion were also used to predict the distribution of these flies three years later in the fall of 2011. In the fall of 2008, eleven years after the first successful release, we estimate that P. tricuspis covered about 50% of the fire ant quarantined area and that it will occur in almost 65% of the quarantine area by 2011. Complete coverage of the fire ant quarantined area will be delayed or limited by this species' slow rate of spread and frequent failure to establish in more northerly portions of the fire ant range and also, perhaps, by its preference for red imported fire ants (S. invicta). Eight years after the first successful release of P. curvatus, two biotypes of this species (one biotype occurring predominantly in the black and hybrid imported fire ants and the other occurring in red imported fire ants) covered almost 60% of the fire ant quarantined area. We estimate these two biotypes will cover almost 90% of the quarantine area by 2011 and 100% by 2012 or 2013. Strategic selection of several distributional gaps for future releases will accelerate complete coverage of quarantine areas. However, some gaps may be best used for the release of additional species of decapitating flies because establishment rates may be higher in areas without competing species.

Distributional patterns of Pseudacteon associated with the Solenopsis saevissima complex in South America.

Classical biological control efforts against imported fire ants have largely involved the use of Pseudacteon parasitoids. To facilitate further exploration for species and population biotypes a database of collection records for Pseudacteon species was organized, including those from the literature and other sources. These data were then used to map the geographical ranges of species associated with the imported fire ants in their native range in South America. In addition, we found geographical range metrics for all species in the genus and related these metrics to latitude and host use. Approximately equal numbers of Pseudacteon species were found in temperate and tropical regions, though the majority of taxa found only in temperate areas were found in the Northern Hemisphere. No significant differences in sizes of geographical ranges were found between Pseudacteon associated with the different host complexes of fire ants despite the much larger and systemic collection effort associated with the S. saevissima host group. The geographical range of the flies was loosely associated with both the number of hosts and the geographical range of their hosts. Pseudacteon with the most extensive ranges had either multiple hosts or hosts with broad distributions. Mean species richnesses of Pseudacteon in locality species assemblages associated with S. saevissima complex ants was 2.8 species, but intensively sampled locations were usually much higher. Possible factors are discussed related to variation in the size of geographical range, and areas in southern South America are outlined that are likely to have been under-explored for Pseudacteon associated with imported fire ants.

Body size, but not cooling rate, affects supercooling points in the red imported fire ant, Solenopsis invicta.

The level of an animal's stress resistance is set by multiple intrinsic physiological and extrinsic environmental parameters. Body size is a critical intrinsic parameter that affects numerous fitness-related organismal traits including fecundity, survival, mating success, and stress resistance. The rate of cooling is a critical extrinsic environmental factor that can affect thermal stress resistance. Workers of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), display considerable variation in adult body size. Therefore, developing ecologically realistic models of thermotolerance in this species requires a consideration of body size. We tested the hypothesis that body size and cooling rate would interact to set the supercooling point in fire ant workers by exposing workers of a range of body sizes to three different cooling regimens: a very fast ramp of -10 degrees C/min, an intermediate ramp of -1 degrees C/min, and an ecologically relevant slow ramp of -0.1 degrees C/min. Specifically, we asked whether large workers were more susceptible to differences in cooling rate than smaller workers. We found that body size had a considerable effect on supercooling point with the largest workers freezing at a temperature approximately 3 degrees C higher than the smallest workers. Cooling rate had a very small effect on supercooling point, and there was no interaction between the two factors. Therefore, the allometry of supercooling points across the range of worker body sizes does not change with cooling rate.

Allomermis solenopsi n. sp. (Nematoda: Mermithidae) parasitising the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) in Argentina.

Allomermis solenopsi n. sp. (Mermithidae: Nematoda) is described from the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) in Argentina. Diagnostic characters of the new species include stiff and erect processes on the surface of the mature egg, small female amphids, extension of the latero-medial rows of male genital papillae beyond the middle rows, an obliquely truncate spicule tip and a ventrally swollen male terminus. This is the first record of Allomermis Steiner, 1924 from South America and the first host record for members of this genus. Previous records of mermithids from Solenopsis spp. are summarised. The placement in Allomermis was confirmed by molecular analyses based on nuclear 18S ribosomal DNA sequences, the first such molecular framework for the Mermithidae. The possible life-cycle of the parasite is discussed, with the aim of using A. solenopsi as a biological control agent for fire ants in the United States.

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.

Female reproductive system of the decapitating fly Pseudacteon wasmanni Schmitz (Diptera: Phoridae).

Pseudacteon wasmanni is a South American decapitating fly that parasitizes workers of Solenopsis fire ants. We used light microscopy (historesin serial-sectioning stained with Haematoxylin/Eosin) and scanning electron microscopy to show and analyze internal and whole external views of the female reproductive system. All specimens analyzed (n=9) by light microscopy showed post-vitellogenic oocytes inside the ovaries. The lack of typical follicles (oocyte-nurse cell complexes) in all specimens suggests that oogenesis occurs during the pupal stage. The total number of eggs found ranged from 31 to 280 (X=142+/-73, SD). The egg has a slugform or torpedo shape (about 130 by 20 microm) with a pointed apex at the posterior pole as defined by the fly; the micropyle appears to be in a depression or invagination at the anterior pole. An acute hypodermic-like ovipositor is evaginated from the hard sclerotized external genitalia during egg laying. The existence of a muscular bulb associated with the end of the common oviduct suggests that the egg is injected into the ant's body by a strong contraction of the bulb which probably is stimulated by bending of several ventral sensilla. During contraction, the abdomen extends out along a large fold between the sixth and seventh tergites in such a way that the sclerotized genitalia is rotated ventrally into a slightly anterior orientation in preparation for oviposition.