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Thelohania solenopsae is a unique microsporidium with a life-cycle finely tuned to parasitizing fire ant colonies. Unlike other microsporidia of social hymenopterans, T. solenopsae infects all castes and stages of the host. Four distinctive spore types are produced: diplokaryotic spores, which develop only in brood (Type 1 DK spores); octets of octospores within sporophorous vesicles, the most prominent spore type in adults but never occurring in brood; Nosema-like diplokaryotic spores (Type 2 DK spores) developing in adults; and megaspores, which occur occasionally in larvae 4, pupae, and adults of all castes but predominantly infect gonads of alates and germinate in inseminated ovaries of queens. Type 2 DK spores function in autoinfection of adipocytes. Proliferation of diplokaryotic meronts in some cells is followed by karyogamy of diplokarya counterparts and meiosis, thereby switching the diplokaryotic sequence to octospore or megaspore development. Megaspores transmit the pathogen transovarially. From the egg to larvae 4, infection is inapparent and can be detected only by PCR. Type 1 DK spore and megaspore sequences are abruptly triggered in larvae 4, the key stage in intra-colony food distribution via trophallaxis, and presumably the central player in horizontal transmission of spores. Molecular, morphological, ultrastructural and life-cycle data indicate that T. solenopsae must be assigned to a new genus. We propose a new combination, Kneallhazia solenopsae.

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.

Sixty four percent of Solenopsis invicta workers infected with Thelohania solenopsis contained 1-6 "cysts" ranging from 70 to 260 microm in diameter. Light and electron microscope analyses showed that cysts are hypertrophied adipocytes transformed by the parasites, each cyst presumably forming from a single cell. In the first step of the pathogenesis, Nosema-like spores functioning in autoinfection are produced; a diplokaryotic sequence leading to their formation causes fat body hypertrophy. When meiosis occurs, it switches parasite development to production of octospores and/or megaspores. Adipocytes become 2-4xlarger than normal in conjunction with intensive parasite multiplication and octospore maturation. Infected cells eventually lose their cellular organization and are converted into reservoirs for spores. There were no manifestations of cellular immunity, such as encapsulation or nodule formation. Similarly, there were no signs of specialized host-parasite interaction that might be interpreted as xenoma-like complexes. The role of the cysts in the parasite's life cycle is unclear. They may represent a defensive reaction of the host sacrificing the infected cells to segregate the infection. Alternatively, the cyst may help protect spores from environmental hazards and provide a concentrated infectious dose to aid horizontal transmission of the microsporidium. We propose to refer to hypertrophied adipocytes filled with T. solenospsae spores as "sporocytosacs", not "cysts."

This is the first report of Thelohania solenopsae infections in monogyne (single-queen) Solenopsis invicta colonies in the field. In a 0.2-ha plot near Baton Rouge, Louisiana, inter-colony prevalence was 63% infection in June, 1999, when the population was 100% monogyne. In February, 2000, 21% of 33 monogyne and 90% of 10 polygyne colonies were infected. By May, 2001, the polygyne colonies had disappeared and only one of 34 monogyne colonies was infected, the final detection of T. solenopsae in the plot. Colony size did not differ significantly among the four types (monogyne versus polygynexinfected versus uninfected).

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8-20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.

The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template. In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.