Vairimorpha invictae Jouvenaz & Ellis (Microsporidia: Burenellidae) is a microsporidian pathogen of Solenopsis fire ants in South America. Because Solenopsis invicta Buren fire ants infected with V. invictae were associated with higher mortality, significantly slower colony growth, and reductions in field populations in Argentina, there is considerable interest in utilizing this pathogen as a classical biological control agent against S. invicta in the U.S. (Briano & Williams 2002; Oi et al. 2005; Briano 2005). V. invictae was formally described by Jouvenaz & Ellis in 1986, however, it was most likely observed much earlier, being the undescribed, dimorphic microsporidium reported from pathogen surveys conducted in Brazil in 1976 and 1979 (Jouvenaz et al., 1980; Briano & Williams 2002). Surveys for V. invictae in Argentina reported prevalence rates of 1, 2.3, and 10% after sampling 1836, 2528, and 2064 nests, respectively (Briano et al. 1995; 2006; Briano & Williams 2002).

In the U.S., there have been surveys for various fire ant pathogens since the 1970s. Limited surveys of red and black imported fire ants, S. invicta and Solenopsis richteri Forel, respectively, were conducted in northern Florida and in Mississippi in 1971 and 1972 (Broome 1974; Jouvenaz et al. 1977). More extensive surveys in the southeastern U.S. of the 2 imported fire ants (S. invicta and S. richteri) and the native fire ants Solenopsis geminata (F.) and Solenopsis xyloni McCook were documented by Jouvenaz et al. (1977). In these surveys, only ubiquitous, nonspecific organisms were detected, with the exception of a fungus that seemed to have minimal effects on S. invicta populations (Broome 1974; Jouvenaz et al. 1977, 1981). In 1989, the fungi Metarhizium anisopliae var. anisopliae (Johnston) Tulloch and a Conidiobolus species were isolated from S. invicta queens collected after a mating flight in Texas (Sánchez-Peña & Thorvilson 1992). Fire ants examined from studies conducted in Alabama, Florida, and Tennessee in 2000–2003 revealed infections of a Neogregarine protozoan, Mattesia sp., and the fungus Myrmicinosporidium durum Hölldobler (Pereira et al. 2002; Pereira 2004) in S. invicta. A microsporidian fire ant pathogen from South America, Kneallhazia (=Thelohania) solenopsae (Knell, Allen & Hazard), was first detected in the U.S. in 1996 (Williams et al. 1998, 2003). Interestingly, subsequent examination of archived S. invicta specimens revealed that K. solenopsae had been present in Texas since the 1980s (Snowden &Vinson 2006). Surveys for K. solenopsae have been conducted in Florida, Texas, Mississippi, and Louisiana, with no reports of V. invictae detection (Pereira et al. 2002; Streett et al. 2004; Mitchell et al. 2006; Milks et al. 2008). However, the focus of these surveys was toward K solenopsae. To date, there have been no reports of V. invictae in the U.S. The occurrence of K. solenopsae in the U.S. permitted removal of the quarantine impediment which has facilitated its utilization against S. invicta (Oi & Valles 2009). Because of the aforementioned interest in V. invictae as a biological control agent, we specifically examined samples of S. invicta for V. invictae to ascertain its presence or absence in the U.S.

S. invicta samples were collected mainly in Florida, supplemented by limited sampling forays in Mississippi, Alabama, Georgia, and South Carolina. Sampling sites located in the Florida panhandle, Alabama, and Mississippi were usually at or near highway rest stops or along secondary and tertiary roads near exits of U.S. Interstate Highway 10. These samples were collected in August 2009. Sampling sites in peninsular Florida and Georgia were located along six roadside transects (1 near Macon, Georgia; 4 near Gainesville, Florida; 1 near Moore Haven, Florida) of about 3.5 miles with stops at roughly 0.5 mile intervals (8 sites in each transect). When possible, 3 representative fire ant nests were sampled at each of the 48 sites in spring 2011. Further sampling occurred in north-central Florida from 100 sites in 5 counties described by Porter (1992). For each of these sites an average of 3 (±1 SD) nest samples were examined. Additional samples (n = 270) were obtained from the Valles et al. (2010) study conducted in the vicinity of Gainesville, Florida (Alachua Co.). Remaining samples were collected at other sites in Florida. The South Carolina sampling was conducted during Sep 2003 at a West Ashley shopping mall on the outskirts of Charleston. This sampling was specifically conducted because one of 4 arbitrarily sampled nests at this location in 1997 revealed a possible observation of V. invictae spores in S. invicta workers (D.H.O. unpublished data).

Adult worker ants were collected by inserting a vial into a S. invicta nest for several minutes which allowed swarming ants to fall into the vial. Vial interiors were coated with talcum powder or fluon (Daikin-Polyflon PTFE D-210, Daikin America, Orangeburg, New York) to prevent ants from escaping. Ants were immediately preserved in 95% ethanol or chilled then frozen alive. Approximately 10–50 ants per nest were examined for V. invictae spores by phase-contrast microscopy or by PCR assay (Valles et al. 2004; Oi et al. 2005). For nests sampled along transects in peninsular Florida and Georgia and in north-central Florida, approximately 10 ants per nest were combined per sampling site and there were usually 3 nests per site. In addition to V. invictae, the presence of K. solenopsae was also documented, and percentages of polygyne nests at some sampling sites were estimated visually or by PCR assay (Oi et al. 2004).

V. invictae was not detected in any of the S. invicta nest samples (Table 1). A total of 1016 samples (i.e., colonies) were examined from 170 sites located in 21 counties among 5 states. The majority of the sampling sites (91%) and nest samples (83%) were from Florida. While social form (i.e. monogyny or polygyny) was not determined for all sites, polygyny was present at 64% of the 114 sites where it was assessed, with an average of 50.6% (±29.8% SD) of the nests being polygyne at the polygynous sites. Polygyny has been associated with higher incidences of pathogen infections in S. invicta (Valles et al. 2010). While it is conceivable that the roadside habitats that were predominantly sampled may not be conducive to V. invictae infections, another fire ant infecting microsporidium, K. solenopsae, was detected in 14.5% (147/1016) of the samples, which is within the 10–31% prevalence reported for K. solenopsae in surveys in Mississippi, Louisiana, and Texas (Streett et al. 2004; Mitchell et al. 2006, Milks et al. 2008). Thus, our methods, which were similar to procedures used to detect both microsporidia in South America (Valles et al. 2004; Briano et al. 1995), should have been sufficient to detect V. invictae in the Florida survey. Furthermore, all PCR reactions included positive controls to verify the proper function of the test. In our search for V. invictae, there is no evidence of this microsporidium being established in S. invicta populations in the U.S. As a result, importation and establishment of V. invictae in U.S. populations of S. invicta remains an important research goal that would add another natural enemy of this invasive ant (Porter 1998).

TABLE 1.

NUMBER OF SOLENOPSIS INVICTA SITES AND NEST SAMPLES PER COUNTY WITH VARIMORPHA INVICTAE AND/OR KNEALLHAZIA SOLENOPSAE.

SUMMARY

Surveys were conducted to search specifically for the microsporidian pathogen Vairimorpha invictae in red imported fire ants (Solenopsis invicta), in the U.S. V. invictae was not detected in any of the 1,016 nest samples collected in 21 counties located in 5 states. A majority of samples (83%) originated from Florida. Despite the absence of V. invictae, another microsporidum, K. solenopsae, was detected in 14.5% of the samples. Based on our sampling, V. invictae is currently not established in the U.S. V. invictae represents an additional natural enemy that could be imported from South America for the classical biological control of S. invicta.