Molecular diversity of the microsporidium Kneallhazia solenopsae reveals an expanded host range among fire ants in North America

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Abstract

Kneallhazia solenopsae is a pathogenic microsporidium that infects the fire ants Solenopsis invicta and Solenopsis richteri in South America and the USA. In this study, we analyzed the prevalence and molecular diversity of K. solenopsae in fire ants from North and South America. We report the first empirical evidence of K. solenopsae infections in the tropical fire ant, Solenopsis geminata, and S. geminata × Solenopsis xyloni hybrids, revealing an expanded host range for this microsporidium. We also analyzed the molecular diversity at the 16S ribosomal RNA gene in K. solenopsae from the ant hosts S. invicta, S. richteri, S. geminata and S. geminata × S. xyloni hybrids from North America, Argentina and Brazil. We found 22 16S haplotypes. One of these haplotypes (WD_1) appears to be widely distributed, and is found in S. invicta from the USA and S. geminata from southern Mexico. Phylogenetic analyses of 16S sequences revealed that K. solenopsae haplotypes fall into one of two major clades that are differentiated by 2–3%. In some cases, multiple K. solenopsae haplotypes per colony were found, suggesting either an incomplete homogenization among gene copies within the 16S gene cluster or multiple K. solenopsae variants simultaneously infecting host colonies.

Introduction

The red imported fire ant, Solenopsis invicta, is an invasive ant that has plagued the southern United States since its introduction in the 1930s from South America. It has spread throughout the southern USA and more recently has been introduced into California. It is also currently found in Australia, Taiwan, mainland China, northern Mexico, and the Caribbean (Buren, 1982, Buckley, 1999, MacKay and Fagerlund, 1997, Davis et al., 2001, McCubbin and Weiner, 2002, Huang et al., 2004, Chen et al., 2006, Sánchez-Peña et al., 2005, Wetterer and Snelling, 2006). S. invicta causes an estimated $6.3 billion in damage annually in urban and agricultural sectors in the USA alone (Lard et al., 2006). In addition, within areas infested by fire ants, nearly 1% of the human population is at risk for anaphylaxis from fire ant stings (Rhoades et al., 1989, Prahlow and Barnard, 1998). Although insecticides are highly effective at controlling S. invicta populations, they must be used on a regular basis to maintain areas free of fire ant populations. Thus, discovery and development of biological control agents remains a high research priority to sustainably control S. invicta.

Kneallhazia solenopsae was originally described as Thelohania solenopsae by Knell et al. (1977) from S. invicta collected in Brazil, but has since been reclassified into the genus Kneallhazia (Sokolova and Fuxa, 2008). K. solenopsae infects all castes and life stages of S. invicta and is transmitted both horizontally and vertically (transovarial transmission) (Briano et al., 1996, Valles et al., 2002, Sokolova and Fuxa, 2008). While the mechanisms of transmission are poorly understood, brood raiding (Tschinkel, 2006) seems to be one pathway for horizontal transmission of K. solenopsae in fire ants (Oi and Williams, 2003). Numerous studies have demonstrated its impact on S. invicta by the debilitation of fire ant queens, resulting in lower weight, reduced fecundity, and premature death (Knell et al., 1977, Williams et al., 1999, Oi and Williams, 2002, Oi and Williams, 2003). Thus, K. solenopsae was identified as a natural enemy with potential for use in the USA against the red imported fire ant.

Few species of microsporidia infect multiple distantly related hosts, and specificity to one host or a related group of hosts is very common (Baker et al., 1995, Vossbrinck et al., 2004, McClymont et al., 2005). K. solenopsae spores have been detected microscopically in the black fire ant, Solenopsis richteri, in South America (Allen and Silveira-Guido, 1974, Briano et al., 1995), in other Solenopsis species from Brazil (Allen and Buren, 1974), and in Solenopsis daguerrei, a social parasite of fire ants (Briano et al., 1996). K. solenopsae spores were not found in other ant genera in South America (Briano et al., 2002), suggesting that the host range of K. solenopsae is restricted to the genus Solenopsis. An extensive survey for fire ant pathogens was conducted in the mid 1970s in the USA (Jouvenaz et al., 1977). Among 1007 colonies of S. invicta examined, a microsporidian infection was present in only one colony. The same microsporidium was found in four of 307 colonies of the tropical fire ant Solenopsis geminata, leading the authors to conclude that S. geminata was the natural host (Jouvenaz et al., 1977). No further description of this microsporidium was provided in the study (Jouvenaz et al., 1977). A more recent survey in 1996 indicated that K. solenopsae was widely distributed in S. invicta populations throughout the southern USA (Williams et al., 1998), and subsequent examination of archived S. invicta samples from Texas revealed K. solenopsae infections in ants dating as far back as 1984 (Snowden and Vinson, 2006). Surveys of other non-S. invicta, field-collected ants, including S. geminata, Dorymyrmex bureni, Pheidole metallescens, Pheidole moerens, Camponotus floridanus, Trachymyrmex septentrionalis, and Brachymyrmex depilis, were all negative for K. solenopsae (Williams et al., 1998).

Recently, in an unpublished report, Snowden and Vinson (2007) described K. solenopsae in S. geminata from Texas. These authors sequenced a portion of the 16S ribosomal RNA gene of K. solenopsae collected from 64 S. invicta and 29 S. geminata colonies, and found a total of 12 16S sequences that differed by 0–5%. Further, some of the sequences were shared between sympatric S. invicta and S. geminata colonies. Another molecular study of K. solenopsae spores isolated from S. invicta from Florida and Brazil, and S. richteri from Argentina showed that the K. solenopsae variant from Florida was genetically distinct but closely related (∼1% base pairs differences) to the microsporidium found in S. invicta from Brazil and S. richteri from Argentina (Mosser et al., 2000). These results suggest an expanded host range for K. solenopsae, as well as high genetic diversity within nominal K. solenopsae. The primary objective of our study was to confirm whether K. solenopsae infects S. geminata. In addition, we conducted comparative phylogenetic analyses of K. solenopsae sequences that were isolated from different Solenopsis hosts in North America, from S. invicta from Brazil and S. richteri from Argentina (Mosser et al., 2000), as well as the microsporidium Kneallhazia sp. from a thief ant, Solenopsis carolinensis.

Section snippets

Collection of ants

Ants were collected from nests, pitfalls, and baits using aspirators or forceps and immediately preserved in 95% ethanol. A total of 450 samples was analyzed; 117 collections were from S. invicta, and 273 samples included S. geminata and S. geminata × Solenopsis xyloni hybrids (Table 1). Collections were obtained in the USA (Florida, South Carolina, Texas), and Mexico (Tamaulipas, Nuevo León and Veracruz) (Table 1, Fig. 1). Taxonomic identification of ants was made by the collectors with

Population screening

Our survey data showed that the prevalence of K. solenopsae varied among sampling sites (Table 1, Fig. 1). Forty-three of the 67 S. invicta colonies from Ocala (Florida) were infected with K. solenopsae and only six of 92 S. invicta colonies from Aiken (South Carolina) were infected. In contrast, none of the 18 S. invicta colonies surveyed from Austin (Texas) were infected. Among all USA S. geminata surveyed, none from Alachua County (Florida) were infected and only a single colony was infected

Discussion

We surveyed the fire ants S. invicta and S. geminata, as well as S. geminata × S. xyloni hybrids for K. solenopsae (or a variant thereof) infections. All species were infected, though the prevalence of infections varied considerably among populations. Although the presence of K. solenopsae in S. geminata has been reported previously (Snowden and Vinson, 2007), this is the first report of this microsporidium in S. geminata × S. xyloni hybrids. This is also the first attempt at describing the genetic

Acknowledgments

We appreciate the technical assistance provided by E. Menas and C. Strong (USDA, ARS, CMAVE), and D. Milne (USDA, ARS, CMAVE) for producing maps. We appreciate the help of Dora Estrada and Amanda Swift in the University of Texas lab. Julian Resasco (University of Florida, UF) provided us with the ants from South Carolina. We wish to thank Matt Gitzendanner (UF, Florida Museum of Natural History) for providing access and help in the use of the UF Genetic Institute Fisher Computer Cluster. We

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