Prevalence and Genetic Diversity of a Microsporidian Parasite in the Black Imported Fire Ant and Its Social Parasitic Ant (Formicidae: Myrmicinae: Solenopsis) in Buenos Aires Province, Argentina

Simple Summary The Black Imported Fire Ant, Solenopsis richteri, was accidentally introduced to the southern U.S. in the 1930s from South America. Long-term, sustained suppression approaches through biological control are needed to control growing invasive fire ant populations. Among the natural enemies used in the U.S., there have been multiple releases of a fungal-like microsporidian pathogen, Kneallhazia solenopsae. In this study, researchers characterized the prevalence and genetic diversity of this microsporidium in native populations of S. richteri. The goal of these types of studies is to assess how the natural enemy acts in the native environment to improve biological control methods. The researchers analyzed ants from 219 nests and found that the microsporidium was present in 12.8% of the nests. Interestingly, within these S. richteri colonies, researchers found a social parasitic ant, Solenopsis daguerrei, and when those ants were analyzed, 3.9% of S. daguerrei ants tested positive for the microsporidium. The microsporidian variants found in both Solenopsis species were genetically similar. Further studies are needed to evaluate the pathogenicity of this microsporidian variant. Abstract Microsporidia are natural pathogens of arthropods and have been used as biological control against insect pests. In the United States, efforts to control the invasive Red Imported Fire Ant, Solenopsis invicta, and Black Imported Fire Ant, Solenopsis richteri, have included the use of the microsporidium, Kneallhazia solenopsae. However, there is limited information about the genetic differences among the microsporidian variants found in S. invicta and in S. richteri. In this study, we assessed the prevalence and genetic diversity of K. solenopsae in native populations of S. richteri in Argentina (South America). Additionally, we examined the social parasitic ant, Solenopsis daguerrei, which is found in some S. richteri nests, for the presence of this microsporidium. The survey of 219 S. richteri nests revealed K. solenopsae infections in all five sites analyzed, with 28 colonies (12.8%) positive for the microsporidium. Among the 180 S. daguerrei individuals collected, seven ants (3.9%) from three sites tested positive for K. solenopsae. Phylogenetic analyses of the microsporidian variants present in S. richteri and S. daguerrei based on partial small subunit ribosomal gene sequences (SSU rRNA) showed that both ant species shared the same variant, which is different from the ones found in S. invicta. Further studies are needed to determine the pathogenicity of genetically different K. solenopsae variants among Solenopsis species.


Introduction
Fire ants belong to the genus Solenopsis, which comprises approximately 190 species of ants [1].Two of these species, the Black Imported Fire Ant, S. richteri (Forel), and the Red Imported Fire Ant, S. invicta (Buren), are native to South America and were accidentally introduced to the southern United States (U.S.) in the 1930s and 1940s [2][3][4].Over the past 90 years, S. invicta has spread extensively, covering much of the southern U.S. and parts of California [5].Notably, S. invicta has also invaded other regions of the world, such as Australia, Taiwan, China, South Korea, Japan, and the Caribbean [6][7][8].On the other hand, invasive populations of S. richteri have been limited to certain areas of the southeastern U.S. where they have been interbreeding with S. invicta, resulting in unique hybrids only found in the U.S. [9].Invasive fire ants are estimated to cost USD 6 billion annually in damage repair and control costs in the U.S. [10].The affected sectors include households, electric and communications systems, agriculture, native species, and human health [10].Integrated pest management for invasive fire ants is evolving but still mainly reliant on insecticides [11].Long-term, sustained approaches through biological control methods are needed to suppress these growing invasive fire ant populations.Significant progress has been made in using natural enemies as biological control agents against invasive fire ants in the U.S.Among those natural enemies, there have been releases of parasitoid phorid flies in the genus Pseudacteon; viruses; and a fire ant microsporidian pathogen, Kneallhazia (=Thelohania) solenopsae Knell, Allen, and Hazard (Microsporidia: Thelohaniidae) [12].
Microsporidia are unicellular fungal parasites that have a broad impact on various animal groups, particularly insects [13].Native populations of S. richteri infected with K. solenopsae experienced an 83% decrease in the number of active colonies [14], while K. solenopsae vegetative stages have been observed in all developmental stages from eggs to adult ants [15].While the detrimental effect of K. solenopsae in S. richteri is well-documented, less is known about the genetic variability of K. solenopsae in S. richteri and whether the K. solenopsae infecting S. invicta and S. richteri are the same or different.Thus, a better knowledge of the genetic diversity of K. solenopsae is needed to improve biological control methods against fire ants.
The sequence diversity from the small subunit ribosomal gene (SSU rRNA) in K. solenopsae was first studied by Moser et al. [16].They analyzed only three K. solenopsae isolates: one isolate from S. invicta in Florida (U.S.), one from Brazil, and one isolate found in S. richteri from Argentina.Despite the limited sampling, the authors found significant genetic variation among the different K. solenopsae isolates and suggested they be considered a species complex (K.solenopsae complex) [16].On the other hand, cross-infection assays between species have shown that inoculations with K. solenopsae isolates from S. invicta from Florida and S. richteri collected in Argentina resulted in 56% fewer infections when inoculations were made to non-conspecific host colonies (Oi, unpublished data).While these reductions in infection could be attributed at least partly to poor cross-fostering of brood inocula, there is still the need to increase our knowledge of the genetic diversity of this microsporidium and the potential link to host specific pathogenesis.
To improve our understanding of the genetic diversity of K. solenopsae among Solenopsis species, Ascunce et al. [17] conducted a second study that included fire ants found in North America, namely S. invicta, S. geminata (the tropical fire ant), and S. geminata × S. xyloni hybrids, revealing an expanded host range for this microsporidium that was found in all the species analyzed [17].Numerous K. solenopsae SSU rRNA variants were found that clustered in two divergent clades [17].Furthermore, some variants were species-specific, while others were shared between S. invicta and native fire ants [17].The study suggested Insects 2023, 14, 901 3 of 10 the possibility of genetically different K. solenopsae species, or, at the very least, different genetic variants that seem to exhibit host preferences.
In this study we assessed the prevalence and genetic diversity of K. solenopsae in S. richteri from South America.Additionally, we included in the analysis the inquiline social parasite, Solenopsis daguerrei (Santschi), which was found parasitizing queens in some S. richteri nests.We hypothesized that the K. solenopsae variants in S. richteri would be different from the ones described in S. invicta.For the K. solenopsae variants in S. daguerrei, we expected those to be the same as the ones in S. richteri due to the intimate relationship between this inquiline social parasite and the ant host, and our previous work suggesting the potential of horizontal transmission among different Solenopsis species [17].

Sample Collection
S. richteri's native distribution extends mostly in higher latitudes in South America compared with S. invicta's range and includes the subtropical grasslands, Mesopotamia and Pampas.For this study, S. richteri worker ants were collected from 219 nests located across five geographic sites in the Province of Buenos Aires, Argentina.In 180 of those 219 S. richteri nests, its inquiline social parasite S. daguerrei was also found.In those cases, a single adult specimen (queen or male) was also assessed for the presence of the microsporidium.Ants were preserved in 95% ethanol.

Molecular Survey
Bulk (~10 worker ants) of S. richteri and individual DNA extractions of S. daguerrei were performed using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MI, USA).DNA concentration was measured using a spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific, Wilmington, DE, USA).Polymerase chain reactions (PCR) were then used to amplify a partial region of the SSU rRNA gene to detect the presence of K. solenopsae following Valles et al. [18,19].The assay is extremely sensitive and will result in successful SSU rRNA K. solenopsae amplification with at least one ant infected among a bulk sample of 10 workers (ratio of 1:10 infected to uninfected worker ants).All PCRs were performed as described in Ascunce et al. [17], and they were accompanied by both a known K. solenopsae positive control and a blank DNA-free negative control.Five µL of the PCR product were loaded onto an agarose gel and subjected to electrophoresis.The gel was stained with ethidium bromide and amplicons were visualized with UV light.

PCR Amplification and Sequencing
PCR amplification to obtain a larger portion of the SSU rRNA gene was conducted on eight of the K. solenopsae-positive samples using K. solenopsae-specific SSU rRNA gene primers: P933 (5 -TAGTATGTTTTGTAAGGGAGAACATAGACTATGACG-3 ) and P935 (5 -ATACGGGACTATAACCCTGTATCGTGTCTGT-3 ).Thermal cycling conditions for amplification reactions conducted using primers P933 and P935 were as follows: One cycle at 94 • C for 2 min, 35 cycles of 94 • C for 15 s, 59 • C for 15 s, 68 • C for 1.5 min, and a final extension cycle at 68 • C for 5 min.In this case, PCRs were conducted in 25-µL reactions containing Platinum Taq (hot start) (Invitrogen, Carlsbad, CA, USA), 0.4 µM of each primer, 0.5-to-1 µL of total genomic DNA (25 to 50 ng) and water.PCR amplicons were purified using magnetic beads (Agencourt AMPure, Beverly, MA, USA) and used in standard fluorescent cycle-sequencing PCR reactions (ABI Prism BigDye TM Terminator chemistry, Applied Biosystems, Foster City, CA, USA).Sequencing reactions were purified using Agencourt CleanSEQ (Beverly, MA, USA) magnetic beads and run on an automatic sequencer at the sequencing core facility (ICBR) at the University of Florida.
The MEGA version X [20] was used to estimate a matrix of pairwise differences among SSU rDNA sequences using uncorrected p-distances (proportion of nucleotide sites at which two sequences being compared are different) as well as the total number of differences.All positions containing gaps and missing data were eliminated (complete deletion option), which led to a total of 760 positions in the final dataset.Neighbor-joining (NJ) trees [21] were generated using the matrix of p-distances.Bootstrapping was performed using 500 pseudoreplications of the dataset.The genealogical relationships among microsporidian variants were also analyzed using the program TCS Version 1.13 [22].

Prevalence of the Microsporidium and the Social Parasite
The social parasite S. daguerrei was found in 180 nests out of the 219 S. richteri nests included in this study representing an average 82% of prevalence.S. daguerrei was found in every sampled S. richteri nest at four sites, and in 12 out of the 51 S. richteri nests.
The K. solenopsae SSU rRNA was successfully PCR amplified from DNA extractions of both ant species, S. richteri and its social parasite, S. daguerrei, indicating that both species harbor this microsporidium.Our PCR assay revealed that the microsporidian prevalence varied among sites and species.For S. richteri, all five sampled sites had some positive colonies, and 28 (12.8%)out of the 219 S. richteri colonies analyzed were positive for K. solenopsae (Table 1).For S. daguerrei, among the 180 inquiline social parasites found in S. richteri colonies, seven ants (3.9%) tested positive for K. solenopsae at only three out of the five sites.This is the first population prevalence report of K. solenopsae in S. daguerrei (Table 1).We observed SSU rRNA PCR-positive amplifications in S. daguerrei in all but one SSU rRNA PCR-positive S. richteri nest.This is that there was one SSU rRNA PCR-positive amplification from a S. daguerrei ant that was found in a S. richteri nest that had a negative PCR (no amplification) result.

Phylogenetic Results
We obtained for the first time the sequence of the K. solenopsae SSU rRNA gene from the social parasite S. daguerrei (Table 2).However, further studies are needed to assess whether the microsporidium causes any type of negative effect on S. daguerrei.Based on the 760 bp alignment, which excluded gaps and missing data, all the K. solenopsae sequences found in S. richteri (host) and S. daguerrei (social parasite) were identical, showing zero differences (Table 3).Furthermore, the sequences were the same as the previously published K. solenopsae found in the S. richteri host (GenBank Accession number: AF031537, Moser et al. [16]), classified as Genotype SA_1 per Ascunce et al. [17].Phylogenetic analyses using the p-distances revealed that the K. solenopsae variant found in S. richteri and S. daguerrei is closely related to the variants found in S. invicta from U.S. and South America (Figures 1 and S1).We constructed a haplotype network using the statistical parsimony method implemented in the program TCS to further understand the relationships among the SSU rRNA sequences (Figure 2).The network method used in this study is based on statistical parsimony of the genealogical relationships among sequences and it included missing and ambiguous data.This network showed that the variant found in S. richteri (host) and S. daguerrei (social parasite) in one of the sites (Bolivar) matched the SA_1 genotype and was differentiated by one mutational step from the sequences obtained from the ants in the other geographic sites (Figure 2).Four of the new SSU rRNA sequences obtained in this study from S. richteri and S. daguerrei from Bolivar are shown in the same node with the SSU rRNA K. solenopsae variant obtained by Moser et al. [16] in S. richteri from Buenos Aires province.The sequence SA_2 corresponded to the SSU rRNA K. solenopsae variant found in S. invicta from Brazil (native range), and it was connected to the new sequences by , and USA_3, in bold are the K. solenopsae variants found among S. richteri: SA_1, Sric_154, Sric_174, Sric_266, and Sric_268, and italicized are the ones found among S. daguerrei ants: Sdag_259, Sdag_268, Sdag_24A and Sdag_24M.This tree was rooted using the WD_1 sequence, which is found in both S. invicta from the USA, as well as S. geminata from southern Mexico, and it belongs to the Neotropical (NeoT) Kneallhazia clade [17].
by three mutational steps.Three, four, and five mutational steps connected SA_2 to the USA_1, USA_2, and USA_3 variants found in S. invicta in the US, respectively.The variant WD_1 that was detected in both S. invicta (US) and S. geminata (Mexico) was the most distant from the new sequences and separated by nine mutational steps.There is a need to improve the resolution of the phylogenetic relationships among these variants by sequencing additional genes.A map with the sampling locations and the geographic distribution of the SSU rRNA K. solenopsae variants is presented in Figure 2.

Discussion
In this study, we surveyed the fire ant S. richteri and its inquiline social parasite S. daguerrei for the presence of the microsporidium K. solenopsae.Although the presence of K. solenopsae in these species has been reported previously [23], this is the first population prevalence report of this microsporidium in S. daguerrei.This is also the first attempt at

Discussion
In this study, we surveyed the fire ant S. richteri and its inquiline social parasite S. daguerrei for the presence of the microsporidium K. solenopsae.Although the presence of K. solenopsae in these species has been reported previously [23], this is the first population prevalence report of this microsporidium in S. daguerrei.This is also the first attempt at characterizing the genetic diversity in this microsporidium among several S. richteri nests from different geographic sites and the microsporidian variants found in S. daguerrei.These SSU rRNA sequence data revealed that (a) K. solenopsae found in S. richteri is very similar (only one mutational step difference detected) among these populations located in Buenos Aires Province, Argentina; (b) K. solenopsae found in the inquiline social parasites were the same as their hosts; and (c) This microsporidian SSU rRNA variant is different from those found among S. invicta populations.Thus, our initial hypothesis and expectation were corroborated based on the current data.

Prevalence
Our survey data showed that K. solenopsae was present in all sites in S. richteri, with a prevalence variation from 5% to 23% of the colonies.The microsporidium was absent in some sites in the social parasite S. daguerrei analyzed, varying from 2% to 13% of the ants in other sites (Table 2).A previous long-term study in one of the sites (Saladillo) found a prevalence of K. solenopsae in S. richteri ranging from 22.4% at the beginning of the study to a final prevalence of 35.7% at the end of the study [14].Interestingly, in this current study, Saladillo had the highest prevalence of K. solenopsae at 19.5%, which is close to one of the values found in the previous study [14].Two differences between that study and this one are the assay used and the number of ants screened; Briano et al. [14] used thousands of ants per nest and detected the microsporidium via microscopy, whereas in our study, we only used 10 ants per nest and conducted the screening using PCR techniques.In another study, Briano et al. [24] also studied the prevalence of K. solenopsae among fire ants in Buenos Aires Province using microscopy.The authors found that some sites had prevalence values below 4%, with a highest value of 27% in one site and an overall average prevalence of 8% [24].Our study builds upon prior research that suggests that K. solenopsae is a common pathogen of fire ants in Buenos Aires Province.

Genetic Identities of Microsporidian Variants
Sequence analyses of the host (S. richteri) and the social parasite (S. daguerrei) showed that both ant species share the same microsporidian variant per geographic site.We hypothesize that due to the higher prevalence of K. solenopsae in S. richteri, this ant is the native host of this microsporidium.In addition, the presence of the same variant in the two studied species likely results from horizontal transfer due to the close relationship between S. richteri and S. daguerrei, such that the pathogen K. solenopsae can be transmitted to S. daguerrei.The ability of this microsporidium to switch among Solenopsis host species has been previously suggested [17].Host switching events have also been observed among the microsporidium Nosema infecting different species of bumblebees [25] and honey bees [26].Interestingly, Nosema ceranae appears to have switched hosts from its native Asian honey bee (Apis cerana) host to the western honey bee (Apis mellifera) in areas where both honey bee species co-occur.In this new host, N. ceranae has a greater virulence than its congener N. apis [26].Thus, it is possible that the lower prevalence of K. solenopsae in S. daguerrei could be due to a higher pathogenicity of this microsporidium in this non-native host.However, Briano et al. [15] presented a different interpretation and considered that the low number of S. daguerrei ants that carried K. solenopsae spores may suggest that S. daguerrei ants have merely ingested spores and were not a real host for the pathogen.Further studies are needed to understand the horizontal transmission patterns of this microsporidium within and among different hosts within a nest and the pathogenicity of K. solenopsae towards S. daguerrei.

Genetic Diversity
The SSU rDNA sequences from S. richteri and S. daguerrei were identical per geographic site.Among all the SSU rRNA sequences from the NeoT clade the percent of pairwise p-distances varied from 0.11 to 0.4% (Table 2).Thus, additional gene sequence data are needed to resolve the phylogenetic relationships among K. solenopsae variants found among the different Solenopsis species, as was previously suggested [17].There is a pressing need to characterize the genome of K. solenopsae as an initial step in unraveling the many aspects of K. solenopsae-fire ant evolutionary interactions.The study of coding genes will also open the door to functional transcriptomic studies to gain a deeper understanding of pathogenicity and transmission modes.

Conclusions
Further studies are needed to gain a better understanding of the evolutionary history of K. solenopsae among fire ants in their native range in South America, as well in their invasive range in the U.S. Gaining a deeper understanding of the genetics and biology of this microsporidium species is crucial for developing effective biological control methods for invasive populations of S. invicta, S. richteri, and its hybrids in the U.S. Determining the pathogenicity (including infectivity and virulence) of genetically different K. solenopsae variants could lead to discovery of new variants that are more effective in controlling fire ants in the U.S.This tri-partite system with the microsporidium Kneallhazia, S. richteri, and its social parasitic ant provides an ideal model for studying the epidemiological dynamics of the microsporidium within a colony.Such knowledge can help to understand

Figure 1 .
Figure 1.Neighbor-joining (NJ) tree constructed based on the K. solenopsae SSU rRNA p-distances found among fire ants.Numbers on branches represent bootstrap support values.Tree based on sequences recovered from K. solenopsae SSU rRNA found among S. invicta genotypes: SA_2, USA_1, USA_2, and USA_3, in bold are the K. solenopsae variants found among S. richteri: SA_1, Sric_154, Sric_174, Sric_266, and Sric_268, and italicized are the ones found among S. daguerrei ants: Sdag_259, Sdag_268, Sdag_24A and Sdag_24M.This tree was rooted using the WD_1 sequence, which is found in both S. invicta from the USA, as well as S. geminata from southern Mexico, and it belongs to the Neotropical (NeoT) Kneallhazia clade[17].

Figure 2 .
Figure 2. The statistical parsimony network and a map showing the distribution of K. solenopsae SSU rRNA variants throughout the Americas indicated by the colored pies.In the network, each connecting branch represents a single mutational step, and inferred missing intermediate haplotypes are represented by white circles.Sizes are scaled and represent relative frequencies.The legend explaining each of the colored pies including the host species, in brackets, is shown in the top right corner of the figure.A detailed of the Province of Buenos Aires, Argentina is shown in the bottom right corner of the figure.

Figure 2 .
Figure 2. The statistical parsimony network and a map showing the distribution of K. solenopsae SSU rRNA variants throughout the Americas indicated by the colored pies.In the network, each connecting branch represents a single mutational step, and inferred missing intermediate haplotypes are represented by white circles.Sizes are scaled and represent relative frequencies.The legend explaining each of the colored pies including the host species, in brackets, is shown in the top right corner of the figure.A detailed of the Province of Buenos Aires, Argentina is shown in the bottom right corner of the figure.

Table 1 .
Prevalence of Kneallhazia solenopsae in S. richteri and its inquiline social parasite, S. daguerrei, in Buenos Aires Province, Argentina.For S. richteri, a bulk sample of 10 ants per nest was analyzed.For those S. richteri nests where S. daguerrei was found, one S. daguerrei ant per S. richteri nest was screened for the presence of K. solenopsae.

Table 2 .
Information of K. solenopsae SSU rRNA sequences, their ant host species, and GenBank accession number information.