Prevalence of Thelohania solenopsae infected Solenopsis invicta newly mated queens within areas of differing social form distributions

Newly mated queens (NMQs) originating from monogyne red imported fire ant (Solenopsis invicta) colonies and following a mating flight, initiate new colonies by sealing themselves in a nuptial chamber and relying solely on their own fat and crop reserves, as well as no longer needed wing muscles to rear their first workers (claustral colony foundation). This method of colony-founding is rarely successful for polygyne-derived NMQs, whose low weight critically limits the number of first workers they are able to produce. However, this observation may be confounded by the parasitic microsporidium, Thelohania solenopsae, thus far found to persist only in association with polygyne colonies. Infections of this microsporidium reduce the weight of female alates and may explain why polygyne NMQs are unlikely to successfully found colonies claustrally. NMQs collected following mating flights in Gainesville and Ocala, Florida were sorted by weight, checked for insemination and T. solenopsae infection. Insemination levels were greater than 90% for all weight classes at both collection sites and were not related to infection. Infection levels were lower in Gainesville than Ocala, averaging 1.67% and 14.14%, respectively. Polygyne-derived NMQs collected in Ocala, defined here as weighing 12mg (social form correctly assigned in 85% of samples examined by PCR), had the highest infection levels, 25.37% (17/67) in 2003 and 21.43% (6/28) in 2004. We conclude that infection by T. solenopsae cannot be completely responsible for the inability of polygyne NMQs to claustrally establish colonies.     

Detection of Thelohania solenopsae (Microsporidia: Thelohaniidae) in Solenopsis invicta (Hymenoptera: Formicidae) by multiplex PCR

Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.     

Effect of mono- and polygyne social forms on transmission and spread of a microsporidium in fire ant populations

Thelohania solenopsae is a pathogen of the red imported fire ant, Solenopsis invicta, which debilitates queens and eventually causes the demise of colonies. Reductions of infected field populations signify its potential usefulness as a biological control agent. Thelohania solenopsae can be transmitted by introducing infected brood into a colony. The social forms of the fire ant, that is, monogyny (single queen per colony) or polygyny (multiple queens per colony), are associated with different behaviors, such as territoriality, that affect the degree of intercolony brood transfer. T. solenopsae was found exclusively in polygyne colonies in Florida. Non-synchronous infections of queens and transovarial transmission favor the persistence and probability of detecting infections in polygynous colonies. However, queens or alates with the monogyne genotype can be infected, and infections in monogyne field colonies have been reported from Louisiana and Argentina. Limited independent colony-founding capability and shorter dispersal of alate queens with the polygyne genotype relative to monogyne alates may facilitate the maintenance of infections in local polygynous populations. Demise of infected monogyne colonies can be twice as fast as in polygyne colonies and favors the pathogen's persistence in polygyne fire ant populations. The social form of the fire ant reflects different physiological and behavioral aspects of the queen and colony that will impact T. solenopsae spread and ultimate usefulness for biological control.     

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.     

Interaction of an entomopathogen with an insect social form: an epizootic of Thelohania solenopsae (Microsporidia) in a population of the red imported fire ant, Solenopsis invicta

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).     

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.     

Heat curing Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae) of a fitness-reducing microsporidium

Laboratory colonies of the predatory mite Metaseiulus occidentalis in Gainesville, FL were found to be infected with an undescribed microsporidium. Experiments were performed to quantify the effect of infection on the fitness of M. occidentalis and to determine if heat treatment can cure mites of the microsporidium. The colonies tested were derived from an isofemale line so that differences in performance could be attributed to the presence of microsporidia. A subcolony of an uninfected isofemale line was infected with the microsporidium by feeding females infected eggs from another colony of M. occidentalis. Infected mites had a shorter mean (+/-SD) female life span (7.4 +/- 2.9 vs. 10.0 +/- 2.8 days), lower mean oviposition (1.6 +/- 0.7 vs. 2.2 +/- 0.4 eggs/day), and a male-biased sex ratio (43 +/- 16% vs. 57 +/- 15% female progeny). The infection was reduced temporarily in colonies initiated from mites that were reared in a growth chamber at 33 degrees C from egg to adult, but healthy colonies only were established from the progeny of the heat-treated adults. These colonies remained free of infection for 10 weeks.     

Comparative effectiveness of light-microscopic techniques and PCR in detecting Thelohania solenopsae (Microsporidia) infections in red imported fire ants (Solenopsis invicta)

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.     

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.     

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.     

The nature of Thelohania solenopsae (Microsporidia) cysts in abdomens of red imported fire ants, Solenopsis invicta

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."     

Distribution,diversity and drivers of blood-borne parasite co-infections in Alaskan bird populations

Avian species are commonly infected by multiple parasites, however few studies have investigated the environmental determinants of the prevalence of co-infection over a large scale. Here we believe that we report the first, detailed ecological study of the prevalence, diversity and co-infections of four avian blood-borne parasite genera: Plasmodium spp., Haemoproteus spp., Leucocytozoon spp. and Trypanosoma spp. We collected blood samples from 47 resident and migratory bird species across a latitudinal gradient in Alaska. From the patterns observed at collection sites, random forest models were used to provide evidence of associations between bioclimatic conditions and the prevalence of parasite co-infection distribution. Molecular screening revealed a higher prevalence of haematozoa (53%) in Alaska than previously reported. Leucocytozoons had the highest diversity, prevalence and prevalence of co-infection. Leucocytozoon prevalence (35%) positively correlated with Trypanosoma prevalence (11%), negatively correlated with Haemoproteus prevalence (14%) and had no correlation with Plasmodium prevalence (7%). We found temperature, precipitation and tree cover to be the primary environmental drivers that show a relationship with the prevalence of co-infection. The results provide insight into the impacts of bioclimatic drivers on parasite ecology and intra-host interactions, and have implications for the study of infectious diseases in rapidly changing environments.     

Epizootiology of Hyalinocysta chapmani (Microsporidia: Thelohaniidae) infections in field populations of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae): a three-year investigation

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae)

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.     

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.     

Morphological and molecular investigations of a microsporidium infecting the European grape vine moth,Lobesia botrana Den. et Schiff., and its taxonomic determination as Cystosporogenes legeri nov. comb

We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.     

Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville

Hippodamia convergens, the convergent lady beetle, is available for aphid control in home gardens and in commercial food production systems throughout the United States and Canada. Beetles received from commercial insectaries for biological control are occasionally infected with a microsporidium. The objective of this study was to describe the pathogen by means of ultrastructure, molecular characterization and tissue pathology. All stages of the microsporidium were in direct contact with the host cell cytoplasm. Early developmental stages were proximal to mature spores and both were observed throughout the tissue sections that were examined. Merogony resulted from binary fission. Early-stage sporoblasts were surrounded by a highly convoluted plasma membrane and contained an electron-dense cytoplasm and diplokaryon. Ovoid to elongated late-stage sporoblasts were surrounded by a relatively complete spore wall. The polar filament, polaroplast, and anchoring disk were readily observed within the cell cytoplasm. Mature spores were typical of terrestrial microsporidia, with a thickened endospore surrounded by a thin exospore. Spores contained well-defined internal structures, including a diplokaryon, lamellar polaroplast and a slightly anisofilar polar filament with 10-14 coils arranged in a single or double row. A prominent indentation was evident at the apical end of the spore wall proximal to the anchoring disk. Aberrant spores were also observed. These had a fully developed endospore and exospore but lacked any discernable internal spore structures, and were, instead, filled with lamellar or vesicular structures. Typical and aberrant spores measured 3.58 ± 0.2 × 2.06 ± 0.2 μm (n = 10) and 3.38 ± 0.8 × 2.13 ± 0.2 μm (n = 10), respectively. Spores were observed in longitudinal muscle surrounding the midgut and within the fat body, Malpighian tubules, pyloric valve epithelium, ventral nerve cord ganglia, muscles and ovaries. The hindgut epithelium was often infected but the connective tissues were rarely invaded. The life cycle and pathology of the microsporidium bears some resemblance to Nosema hippodamiae, the only microsporidium reported from H. convergens by Lipa and Steinhaus in 1959. Molecular characterization of the pathogen genomic DNA revealed that it is 99% similar to Tubulinosema acridophagus and T. ratisbonensis, two pathogens that infect Drosophila melanogaster and 98% similar to T. kingi from D. willistoni. Based on similarities in pathogen ultrastructure and the molecular information gained during this study, we propose that the microsporidium in H. convergens be given the name Tubulinosema hippodamiae.     

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.