Southeastern Naturalist 83 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 22001166 SOUTHEASTERN NATURALIST V1o5l(.1 1):58,3 N–1o0. 11 Bacteria Associated with Red Imported Fire Ants (Solenopsis invicta) from Mounds in Mississippi Sandra Woolfolk1,2, C. Elizabeth Stokes1, Clarence Watson3, Richard Brown1, and Richard Baird1,* Abstract - A study was conducted to determine microbial community structure and baseline information of cultural bacteria taxa within Solenopsis invicta (Red Imported Fire Ant) mounds from 3 locations along the roadside of Natchez Trace Parkway in Mississippi. At each location, samples consisting of mound soils, plant debris of primarily grass stem and leaves (control), and ant body tissues were obtained from replicate mounds during March, July, and November 2004. Bacteria isolate frequencies from soil were significantly greater than from plant or ant body tissues. Using 16S sequence data, 68 taxa from 2324 isolates were obtained from the 3 substrate types. The 7 most common bacteria following in order of greatest isolation frequencies were Bacillus sp. (5) (species complex), Achromobacter xylosoxidans, Bacillus cereus (complex), Lysininibacillus boronitolerans, Serratia liquefaciens, Pseudomonas protegens, and Lysinibacillus sphaericus. Richness, diversity, and evenness values varied between the locations, sampling dates, and the 3 isolation substrates. Total community-coefficient values were 0.74 to 0.84 across sampling dates. Overall these values indicated uniform communities across the different locations, isolation substrates, and across 3 sampling dates. Furthermore, no consistent trends in frequencies were observed by comparing ant tissues, location, and sampling dates to occurrences of bacterial taxa. Isolates and data obtained from this survey will allow for further testing to determine their role as food sources, saprophytes, or pathogens in Red Imported Fire Ant mound ecosystems. Introduction The history and economic impact of introduction and expansion of the 2 imported fire ants, Solenopsis richteri Buren (Black Imported Fire Ant [BIFA]) and S. invicta Buren (Red Imported Fire Ant [RIFA]), in the United States have been well documented (Lard et al. 2002, Tschinkel 2005). In Mississippi, RIFA has spread from the Gulf Coast northward to the middle part of the state where this species has displaced BIFA or hybridized with it, restricting BIFA to the northeastern and north-central area of the state (Streett et al. 2006). A similar replacement of BIFA with the hybrid species also has occurred in Alabama and Georgia (Tschinkel 2005). RIFA were reported to cause $750 million in losses to agricultural crops and livestock (MacDonald 2006). In rural habitats, imported fire ants have a major impact on ground-nesting animals including soil-inhabiting arthropods, reptiles, birds, and mammals (Lockley 1996, Vinson 1994). With the current lack of knowledge 1BCH-EPP Dept., Box 9655, Mississippi State University, Mississippi State, MS 39762. 2Currnet address - Valent BioSciences, 2142 350th Street, Osage, IA 50461. 3University of Arkansas Division of Agriculture, 2404 North University Avenue, Little Rock, AR 72207. *Corresponding author - rbaird@plantpath.msstate.edu. Manuscript Editor: Glen Mittelhauser Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 84 regarding the occurrence of biological control organisms such as bacteria that might limit their spread, documenting associated microbes (isolation) may be valuable for future management of the fire ants. Complex and diverse microbial communities have been known to inhabit soil (Barron 1972, Nakatsu 2007, Zak et al. 2003) in comparison to a reduced diversity in insect tissues (Peloquin and Greenberg 2003). However, a diverse microflora is harbored in internal body parts of insects (Bignell 1984), especially endosymbiotic microbes in the gut (Douglas 1998, Frederick and Caesar 2000). These symbiotic bacteria can be acquired from the soil environment (Kikuchi et al. 2007) or transmitted vertically (Douglas 1998). This mutualistic relationship has a nutritional basis in that the bacteria serve as a direct food source or provide nutrients unavailable to the insect. Conversely, research on Formicidae showed that some ant species can filter or remove fungi by licking potential pathogens or threats from colonies (Chapman 1998) More recently, ants were reported to physically remove (via grooming) spores or microbial contaminants from the outside of bodies of others to prevent increased infections, spread, and sporulation potential (Jabr 2012). It is possible then that important biological control organisms are being excluded by the ants and could be artificially introduced into mounds to overcome the defenses by the ants. Obtaining isolates of bacteria for testing their role in the mound ecosystem could reveal important data. Many microorganisms have been studied as potential biological control agents that have potential to adversely affect RIFA. Selected microorganisms associated with RIFA and other ants have been surveyed in various regions of the United States (Beckham et al. 1982, Jouvenaz et al. 1977, Zettler et al. 2002). In a past survey, 58 cultural-dependent bacterial taxa were isolated from soils of BIFA and S. invicta x richteri mounds and plant debris within the mounds from several selected counties of northeast Mississippi (Baird et al. 2007). Ishak et al. (2011) compared bacterial associates of workers, brood, and soils in mounds of RIFA and the native S. geminata (Fabricius) (Fire Ant) in Texas. These authors listed 28 bacterial genera commonly associated with workers and brood of RIFA using 454 pyrosequencing data (>1.0% of total bacteria). However, no isolates were obtained from these studies to confirm identities of the 28 genera or to allow for further testing of their associations with the ants. The objective of our study was to obtain baseline data on microbial community overlap and distributions of culturable bacteria (e.g., mutualists, saprophytes, or pathogens) associated with workers and mounds of RIFA in areas where the ants were newly established along the Natchez Trace Parkway in Mississippi. Species richness, diversity, evenness, and community coefficients of the microbes associated with RIFA soils, ant bodies, and plant debris within mounds were also determined. Methods In March, July, and November of 2004, we collected soil samples from 5 ant mounds at 3 locations along the Natchez Trace Parkway: Hinds (mile markers Southeastern Naturalist 85 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 83–87), Madison (mile markers 102–122), and Leake (mile markers 129–138) counties, MS. Overall, the soils were similar in composition and nutrient base, and were well drained to 15 cm in depth. In regard to mound characteristics, the 3 collecting sites had different soil types based on soil survey reports. The Hinds County site consisted of Loring-Memphis soils (fine-silty, mixed, thermic Typic Fragiudalfs and fine-silty, mixed, thermic Typic Hapludalfs; Cole et al. 1979). The Madison County site possessed Byram-Loring soils (fine-silty, mixed, thermic Typic Fragiudalfs) and Providence-Smithdale soils (a mixture of fine-silty, mixed, thermic Typic Fragiudalfs and fine-loamy, siliceous, thermic Typic Paleudults) (Scott et al. 1984), while the Leake County site had Providence-Smithdale soils (a mixture of fine-silty, mixed, active, thermic Oxyaquic Fragiudalfs and fine-loamy, siliceous, subactive, thermic Typic Hapludults) and Smithdale-Providence soils (a mixture of fine-loamy, siliceous, subactive, thermic Typic Hapludults and fine-silty, mixed, active, thermic Oxyaquic Fragiudalfs) (Brass et al. 2009). Mounds were 10 to 15 m from the roadway and primarily in grassy areas. During each month, we sampled 5 active mounds at each location using a shovel to collect 2000 ml of soil from the lower third of the mound. Due to disruption from the previous month’s sampling, we chose new mounds within each area each month. Each sample, stored in plastic bags, contained soil, plant debris (grass stems and leaf tissues), and ants. Between sampling of each mound, we cleaned the shovel by rinsing it in 10% bleach solution for 1 minute followed with tap water for 1 minute. The bags containing the samples were sealed, cooled, and transported to the laboratory and stored for a maximum of 24 hours at 4 °C. From each bag, we used 500-sg subsamples of the soil/plant debris/ants mixture for microbiological assessment. In addition, we collected 20 worker ants from each mound and preserved them in 70% ethyl alcohol for identification (Woolfolk et al. 2004, Baird et al. 2007). Approximately 100–200 worker ants from the 2000-ml samples collected from each mound were also collected and stored in vials that were frozen at -20 °C for either isolation studies of bacteria within 24 hr of sampling or for later gas chromatograph-mass ppectrommetry (GC-MS) confirmations. To verify their identification, we immersed the stored frozen RIFA worker ants in hexane for a minimum of 2 days and then removed and placed the solvent in 2-ml automatic sampler vials. Samples were analyzed using gas chromatograph/mass spectrophotometry (GC-MS) as described by Menzel et al. (2008), and their venom alkaloids and cuticular hydrocarbons were determined at the Biological Control of Pests Research Unit, USDA-ARS, Stoneville, MS. Results from the analyzes verified the species as RIFA. We used trypticase soy agar (TSA; Difco®, Detroit, MI) as the medium for isolating bacteria from the 3 substrates. The TSA was modified with 50 mg/L Nystain (Sigma, St. Louis, MO) to inhibit fungal growth. Bacteria isolation from plant tissue. We removed plant debris from each 500-g soil subsample for further processing and randomly selected 4 pieces of plant tissue (up to 10 cm long) to section into 1-cm2 pieces. We surface sterilized plant tissues using sodium hypochlorite (w/v 0.534) for 30 seconds, and then aseptically placed Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 86 2 of the 1-cm2 pieces into each of 4 TSA plates for a total of 8 pieces from each mound. We incubated the plates at room temperature for up to 1 week. All bacteria growing from the tissues were subcultured onto the TSA. Bacteria isolated from mound soils. We identified bacteria using soil serial dilution procedures as described by Baird et al. (2007). We prepared homogenates of soil samples by adding 1.0 g of soil from each mound into 9.0 ml of sterile distilled water to create a tenfold dilution series up to 10-3 using the methods modified from Baird et al. (2007). Aliquots of 100 ml of the soil homogenate per dilution were pipetted and spread onto the surface of the TSA using sterile, polypropylene bacteria cell spreaders. We used 4 replicate TSA plates/dilution 10-3 for each of the 5 mounds, 3 sampling dates, and 3 locations for a total of 180 samples. Bacteria were subcultured for up to 1 week and stored at room temperature until further processed for identification. Bacteria isolated from ant bodies (external tissues): The sampling consisted of 5 mounds × 3 locations × 3 dates × 4 replicate ants/mound = 180. For processing and isolating bacteria from the external body region of the ants, we initially slowed or immobilized RIFA workers without killing them by placing them in plastic bags at -20 °C for approximately 30 minutes. Two workers were randomly selected from each sample bag (each from a single mound) and placed on a TSA plate by sliding them onto the agar. We prepared 4 replicate plates for each of the 5 mounds to provide 40 worker ants for each of the 3 locations and 3 sampling dates. Thus, we plated a total of of 360 worker ants onto TSA during the study. Following plating, we incubated bacterial cultures at 30 °C for 72 hours and subcultured on TSA all bacteria growing from the ant bodies to obtain pure cultures. Bacteria isolated from ant bodies (internal tissues): Again, the sampling consisted of 5 mounds × 3 locations × 3 dates × 4 replicate ants/mound = 180. For bacterial isolations from the internal body region, we paralyzed 4 randomly selected workers using the freezer method described above for external tissue samples. Each ant was submerged in 1% sodium hypochlorite containing 0.01% Tween-80 (Sigma, St. Louis, MO) for one minute, then submerged in 1% sodium thiosulfite to neutralize the sodium hypochlorite and rinsed twice with sterile distilled water. We chilled the RIFA specimens in sterile 50-mM phosphate buffer containing 0.01% Tween-80 (buffer-Tween). Each ant was placed in a 1.5-ml sterile microcentrifuge tube containing chilled buffer-Tween, ground, and homogenized using a micropestle. We diluted homogenates in tenfold dilution series similar to ones used for mound soils, and spread aliquots of 100 μl from each dilution onto the TSA media using a sterile polypropylene spreader. To obtain pure cultures, we subcultered all bacteria growing from the TSA for up to 7 days after initial plating onto TSA. We used various bacterial identification methods. During the study, we initially grouped morphologically similar isolates of all bacteria. We further tested a minimum of 20% of the isolates from each group to ensure the taxonomic groupings were correct, using 3 methods of identification in the following order: cellular fatty acid methyl esters analysis (fatty acid profiles) by gas chromatography (GC-FAME; Microbial Identification System Inc. [MIDI], Newark, DE) profiles, biochemical Southeastern Naturalist 87 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 tests, and 16S molecular sequence methods as described below. Following molecular identification as the final characterization step, we stored a minimum of 2 representatives of each taxon in 10% glycerol in a 1.2-ml sterile cryogenic vial at -80 °C for permanent culture collection. We performed the GC-FAME analysis according to the manufacturer’s standard protocol for the bacterial community (Gitaitas and Beaver 1990, Peloquin and Greenberg 2003, Sasser 2001, Tighe et al. 2000) and protocol used by Baird et al. (2007) for aerobic bacteria. Samples were run on the Hewlett- Packard 6890 GC automatic liquid sampler (Hewlett Packard, Pittsburg, PA) to obtain fatty acid compositions that were matched against a library of known species (Sasser 2001, Kunitzky et al. 2006). Following the FAME’s (MIDI system) analysis of all the isolates characterized as named taxa (e.g., genus or species) from the library, we further verified isolates per taxonomic grouping using biochemical data. We cultured these isolates on TSA for 24 hours at 30 ºC and conducted biochemical testing using Biolog Identification System (Biolog, Inc., Hayward, CA) according to standard bacteriology references (Brenner et al. 2004, Euzeby 1997, Garrity et al. 2001, Holt et al. 1994, Jouvenaz et al. 1996). These standard biochemical tests included gram reaction, cellular morphology, indole, catalase, cytochrome oxidase, and carbon-source utilization tests. We determined preliminary taxonomic names based on the test results and morphology. We further refinced the grouping of the isolates following the chemical tes ts. The final identification method employed 16S molecular sequence data to confirm identities of isolates with the groups as described by Woolfolk and Inglis (2004). We followed the protocol for extracting gram-positive bacteria with Qiagen DNeasy® Blood and Tissue Kit (Qiagen, Chatsworth, CA). We used genomic DNA obtained from bacteria for final identification of bacterial taxa through PCR procedures to amplify 16S ribosomal RNA (rRNA) genes (Amann et al. 1994, Coplin and Kado 2001), and utilized a GoTaq® PCR Core System I (Promega Corporation, Madison, WI) kit as recommended by the manufacturer. We used negative controls, which contained no DNA templates, in every reaction to check for contamination. PCR products were purified with QIAquick PCR Purification Kit (Qiagen), following procedures recommended by the manufacturer, and sequenced by Eurofins MWG Operon (Huntsville, AL). We visually inspected sequencing data for sequencing errors using the CEQ®8000 Genetic Analysis Software (Beckman Coulter, Fullerton, CA). We constructed the contigs of the sequences using SeqMan of Lasergene version 7.0 software (DNASTAR, Inc., Madison, WI) and then compared sequence data with the GenBank database through BLAST (Basic Local Alignment Search Tool) to determine identities using the blastn program. We deposited sequence data in GenBank (National Center for Biotechnology Information, NCBI). When a sample with 70% coverage or higher within 16S rRNA sequences shared a minimum of 97% of identity with GenBank data, we assumed that the sample was the same species (Claridge 2004, Cohan 2002, Stackebrandt and Goebel 1994). Statistical analyses We calculated relative frequencies of bacterial occurrence and computed the following biodiversity indices: species or taxon richness, Shannon-Weaver species Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 88 diversity index (H´), coefficient of community (CC), and species or taxon evenness (E) (Price 1997, Stephenson 1989, Stephenson et al. 2004). We further analyzed the data with one-way analysis of variance (ANOVA) using the general linear models GLM procedure of SAS® (SAS Institute 1999), and performed Fisher’s protected least significant difference test (P < 0.05) to compare means. Results We identified a total of 68 bacterial taxa from 2324 isolates from external and internal bodies of RIFA, mound soils, and plant debris within the mounds (Appendix 1). The most common genus, Bacillus, consisted of 16 species and comprised almost half the total percentage of all isolations. None of the traditional bacterial or molecular identification methods from this study provide specific epithets for Bacillus sp. 1 to 8 (species or subspecies complex). Using the 16S data, the highest percent isolation frequencies across the study were Bacillus sp. (5) (29.4%), Achromobacter xylosoxidans (9.0%), Bacillus cereus 2 (6.4%), Lysininibacillus boronitolerans (6.3%), Serratia liquefaciens (6.3%), Pseudomonas protegens (6.2%), and Lysinibacillus sphaericus (6.1%) (Appendix 1). In addition, 21 of 68 taxa observed in this study were not isolated from plant debris. The remaining 47 taxa observed from plant debris were generally found at lower percentages than in mounds or ant tissue. Based on MIDI FAME’s profiles, we identified the bacterial species listed above either as unknowns or different taxa compared with 16S sequence data. For initial separating or screening of the large number of isolates obtained, fatty acid data profiles and biochemical data were very reliable in terms of consistence with morphological comparisons, and for pooling isolates into distinct taxonomic groupings. However, differences in species identifications using fatty acid profiles were not consistent with 16S data. For example, fatty acid profiles identified what 16S data indicated was Bacillus sp. (5), which had the highest percent isolation frequencies for bacteria, as Bacillus thuringiensis with a similarity index (SI) = 0.758, Pseudomonas protegens (16S) as Pseudomonas putida biotype A with SI = 0.796, and Lysinibacillus sphaericus (16S) as Bacillus sphaericus Meyer and Neide with SI = 0.700. These results show the inconsistency of MIDI FAME’s acid profiles compared with the 16S data. Species richness values were similar across sampling dates but varied depending upon tissue types and collection locations. Overall, the highest species richness values were from mound soil substrate (61), the Hinds location (60), and March sampling date (65), respectively (Table 1). There were significant differences in values across locations, substrates, and sampling dates. When external and internal bodies of ants were compared, richness values were similar for external (43) and internal bodies (40). Total species richness values for bacteria from mounds were always numerically greater compared with ant tissue and plant debris. Overall bacterial species H´ was 3.45 across all pooled bacterial isolates data. When H´ values between external and internal tissues of the ants’ bodies were compared, values for the internal body tissues indicated that there were a significantly greater number of taxa than the external body tissues at 3.17 and 2.89, respectively. Using comparisons between locations and substrate types, no Southeastern Naturalist 89 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 significant differences in diversity were noted between locations and mound soil or ant tissue, whereas values for plant debris were significantly lower from Madison County than the other two counties (Table 2). Total bacterial species E was 0.81, indicating high relative abundance with most of the isolations during the study belonging to similar species equally across the study sites (Table 3). Specifically, ant tissue E values (mean = 0.82) had significantly greater abundance values than the other 2 substrates. Furthermore, mound data from Madison County (0.80) were significantly lower than that from Hinds (0.83) and Leake (0.85) counties. In addition, E values were significantly lower Table 1. Species richness of all bacterial taxa isolated from Red Imported Fire Ants and mounds along Natchez Trace Parkway in Mississippi, 2004. Within-column values with the same lowercase letter are not significantly different (P > 0.05). Across-row mean values with the same uppercase letter are not significantly different (P > 0.05). Means were compared according to Fisher’s protected least significant difference test (t-test; P > 0.05). Species Species Species Substrate richness Location richness Sampling date richness Mound soil 61 a Hinds 60 a March 65 a Plant debris 45 b Leake 57 ab July 56 b Ant tissue 56 a Madison 52 b November 40 c LSD (P ≤ 0.05) 5.0 5.4 5.0 Species Richness Location Soil Plant debris Ant tissue LSD (P ≤ 0.05) Hinds 46 a (A) 29 a (B) 45 a (A) (10) Leake 44 a (A) 24 a (B) 32 a (B) (10) Madison 42 a (A) 17 b (B) 36 a (A) (6) LSD (P ≤ 0.05) 10 7 9 Table 2. Species diversity (H´) of all bacterial taxa isolated from Red Imported Fire Ants and mounds along Natchez Trace Parkway in Mississippi, 2004. Within-column values with the same lowercase letter are not significantly different (P > 0.05). Across-row mean values with the same uppercase letter are not significantly different (P > 0.05). Means were compared according to Fisher’s protected least significant difference (LSD) test (t-test; P > 0.05). Substrate H´ Location H´ Sampling date H´ Mound soil 3.10 b Hinds 3.41 a March 3.25 b Plant debris 3.02 b Leake 3.43 a July 3.46 a Ant tissue 3.27 a Madison 3.18 b November 3.23 b LSD (P ≤ 0.05) 0.10 0.22 0.15 H´ Location Soil mound Plant debris Ant tissue LSD (P ≤ 0.05) Hinds 2.94 a (AB) 2.81 a (B) 3.19 a (A) ( 0.30) Leake 3.06 a (A) 2.81 a (A) 2.90 a (A) (0.30) Madison 2.88 a (A) 2.20 b (B) 3.08 a (A) (0.43) LSD (P ≤ 0.05) 0.31 0.45 0.25 Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 90 during March samplings (mean = 0.78), compared with the other 2 dates (0.86 and 0.88 for July and November, respectively). Coefficient community values calculated based on data from substrate, location, and sampling date ranged from 0.74 to 0.89 (Table 4). By substrates, the highest CC value was from soil mounds–ant tissue (0.82), and for locations Hinds–Madison (0.89). By sampling dates, the highest value occurred for March–July (0.84). The CC values were also compared on the basis of location and 2 substrates interactions Table 4. Coefficient of community (CC) of all bacterial taxa isolated from Red Imported Fire Ants and mounds along Natchez Trace Parkway in Mississippi, 2004. Substrates CC LocationsA CC Sampling datesB CC Mound soil–Plant debris 0.75 H–L 0.82 Mar–July 0.84 Mound soil–Ant tissue 0.82 H–M 0.89 Mar–Nov 0.74 Plant debris–Ant tissue 0.75 L–M 0.75 July–Nov 0.77 Location Substrates CC Hinds Mound soil–Plant debris 0.69 Hinds Mound soil–Ant tissue 0.68 Hinds Plant debris–Ant tissue 0.62 Leake Mound soil–Plant debris 0.50 Leake Mound soil–Ant tissue 0.61 Leake Plant debris–Ant tissue 0.57 Madison Mound soil–Plant debris 0.51 Madison Mound soil–Ant tissue 0.72 Madison Plant debris–Ant tissue 0.42 A H = Hinds County, L = Leake County, M = Madison County. B Mar = March, Nov = November. Table 3. Species evenness (E) of all bacterial taxa isolated from Red Imported Fire Ants and mounds along Natchez Trace Parkway in Mississippi, 2004. Within-column values with the same lowercase letter are not significantly different (P > 0.05). Within-column values with the same letter are not significantly different (P > 0.05). Across-row mean values with the same uppercase letter are not significantly different (P > 0.05). Means for evenness values were compared according to Fisher’s protected least significant difference test (t-test; P > 0.05). Substrate E Location E Sampling date E Mound soil 0.75 c Hinds 0.83 a March 0.78 b Plant debris 0.79 b Leake 0.85 a July 0.86 a Ant tissue 0.82 a Madison 0.80 b November 0.88 a LSD (P ≤ 0.05) 0.02 0.02 0.04 E Location Soil mound Plant debris Ant tissue LSD (P ≤ 0.05) Hinds 0.77 a (B) 0.84 a (A) 0.84 a (A) (0.05) Leake 0.81 a (B) 0.88 a (A) 0.84 a (AB) (0.05) Madison 0.77 a (B) 0.79 a (B) 0.86 a (A) (0.10) LSD (P ≤ 0.05) 0.06 0.09 0.04 Southeastern Naturalist 91 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 (Table 4). For each location, CC values ranged between 0.50 to 0.69, 0.61 to 0.72, and 0.42 to 0.62, when comparing substrate types mound soil–plant debris, mound soil–ant tissue, and plant debris–ant tissue, respectively. Ant microorganism CC values for external and internal tissues of ants by individual location were compared with the highest value from Madison County (0.86) compared with Hinds (0.47) and Leake (0.44) counties (data not shown). These data further indicate that external and internal tissues of ants from Madison County had similar species of bacteria. Discussion The study was conducted along the Natchez Trace Parkway due to the uniquely protected ecological zone where fire ants became established and spread along this natural corridor. Site data was obtained from soil survey reports, published by the Natural Resources Conservation Service in each county. Since soil nutrients, drainage, and pH were similar across the 3 locations, these environments would not be expected to affect microbial population richness, evenness, and diversity. The mounds were along the roadside within 10 to 15 m of the roadway. The grassy borders are preferred areas where S. invicta become established within this protected natural corridor. Other than periodic mowing along the roadside, no other management practices were employed in these areas that could have impacted the RIFA or microbial communities. This investigation is the first attempt to understand the bacterial communities associated with RIFA species in mounds along the roadsides of Natchez Trace Parkway. Collection and development of a microbial isolate library from RIFA mounds were obtained to enable future studies. Understanding microbial community structure in mounds may lead to a better understanding of the role bacteria and even fungi may have with the ants. However, of particular interest is that many bacterial species could be antagonistic or parasitic to ants and have biological control potentials. The number of taxa from the current investigation are based on culturable bacteria. The taxa were greater in number compared to a previous investigation (58 taxa) from black/hybrid imported fire ants (BIFA and S. invicta x richteri) mounds along roadways in northeastern region of Mississippi (Baird et al. 2007) It was shown that the bacterial community collected at the specific level varied between RIFA in the current investigation and black/hybrid imported fire ants study of Baird et al. (2007). In this previous study, 5 species identified using MIDI FAME’s results (0.71–0.87 SI) had the same names as those provided by the 16S data in GenBank database. The MIDI FAME’s results presented in the previous paper were not confirmed by DNA sequence information, thus making comparisons to this current study unclear. Common species in the earlier study were Chrysobacterium indologenes (Yabuuchi et al.) Vandamme et al., Stenotrophomonas maltophilia (Hugh) Palleroni and Bradbury, Actinomadura yumaensis (Actinomycete) Labeda et al., and Arcanobacterium haemolyticum McLean et al. In a study conducted in Texas, bacterial taxa were obtained from RIFA tissues including mound workers, brood, Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 92 and soil using the newer 454-sequencing methods (Ishak et al. 2011). Only 5 genera in that study were common to the current Mississippi investigation and no specieslevel data was presented from 454 data. The results from Ishak et al. (2011) leaves in doubt what taxa at specific level were involved, especially since no isolates were obtained to verify the data or that from other interaction studies. Compared to numerous studies of other insect species endosymbionts, data from RIFA are limited. In the current study, 42 culturable taxa were identified from internal tissues, with Serratia liquefaciens being the most common. In a study surveying the endosymbiotic bacteria in the midgut of fourth-instar reproductive RIFA larvae, 6 bacterial species were cultured (Medina 2010, Peloquin and Greenberg 2003). The similarities between the taxa were limited to the generic level (Enterococcus and Staphylococcus) in the earlier study. Using 16S data, Medina (2010) identified 10 bacterial taxa isolated from internal tissues, with 2 overlapping in this study (Achromobacter xylosoxidans and Serratia marcenscens). Sequencing using the 16S rRNA gene region data has emerged as the most accurate method of identification compared to other taxonomic tools (Tshikudo et al. 2013). Those authors concluded that 16S sequence data could identify a broader group of bacterial species, including rarely culturable isolates and phenotypical strains of bacteria that could not be identified using other systems such as MIDI FAME analysis and Biolog System. Contrary to the findings of Tshikudo et al. (2013), it was determined that the inability to use 16S to define species from recently divergent relatives makes the other systems (e.g., MIDI FAME and Biolog) valuable for assessing different species complexes (Enright et al. 1994, Fox et al. 1992). Since our study was primarily concerned with community or bacterial populations, we employed 16S sequence data for species determinations. We retained the isolates of culturable bacteria for future research to determine their role or possible function in mounds and as biological control agents of RIFA. In this investigation, we identified 3 strains of Serratia marcescens, a species considered to be a facultative pathogen (Bucher 1960, 1963) that can cause death in different insect species if the bacteria enter the hemocoel (Tanada and Kaya 1993). This bacterial species might be a potential pathogen for control of imported fire ants, but future in situ studies are needed to confirm this hypothesis. Another species found in our study, Achromobacter xylosoxidans, was previously reported from aquatic environments and is pathogenic to humans with immunodeficiencies (Duggan et al. 1996), but the pathogenicity of this species to RIFA is unknown. Approximately two-thirds of the taxa associated with plant debris were also present in mounds and ant tissue, indicating that their role might be as saprobes and not directly associated with the ants, only occurring in ant tissue and mound by way of incidental transmission from the immediate surroundings. (Appendix 1). In a previous study of bacteria on black/hybrid imported fire ants, 51 of 58 taxa isolated did not occur on the plant debris (Baird et al. 2007). Total species richness from mound soils were always greatest compared to the plant debris and ant bodies by locations and dates in our study. These results are similar to the previous investigation by Baird et al.(2007). In that study, a thorough Southeastern Naturalist 93 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 discussion of mound soils and the reasons they harbor diverse and greater populations of microbes are reviewed. Nutrient diversity in soils contributes to a greater diversity of microbes (Barron 1972) as compared to plant tissues and ant bodies. Total species diversity values for bacteria showed slightly different trends from species richness. The highest total species diversity values for bacteria were from ant tissue. This finding indicates that the the relative significance of individual species found in proportion of the total numbers of different species was greatest on the ants themselves. In addition, it was interesting to note that diversity values found in external and internal bodies of ants were similar for RIFA in our study, but overlap of these species was approximately 60% between the tissue types. Results of previous studies (Baird et al. 2007) differed from the current investigation, in that the highest bacterial diversity values were found in mound soil harboring black/hybrids. This discrepency may be due to a difference in sampling dates, ant species, or sample-location ecological characteristics between the 2 studies. Das et al. (2007) suggested that microbial taxa can have different ecological roles from one location to another, which at certain times may influence the diversity values as well as impact microbial community structural differences. Evenness and CC values are an important indicator in measuring biodiversity within a community of taxa since they take into account relative abundance compared with species richness. The values for E shown in Table 3 were 0.74 or greater, indicating that the majority of species were common to both communities being compared. The lower evenness values for March compared with the other 2 sampling dates may be an indication of temperature and rainfall differences versus actual population numbers of bacterial taxa. However, precipitation and temperatures were generally consistent among the location during the 3 sampling dates. A recent study reported that initial community evenness was a key factor in preserving the functional stability of an ecosystem (Wittebolle et al. 2009). In that investigation, denitrifying bacterial communities were compared in microcosms where both richness and initial evenness values were measured. The study reported that the stability of the net ecosystem denitrification in the face of salinity stress was strongly affected by the initial evenness of the community. When one species is extremely dominant within the community (highly uneven), the population is less resistant to environmental stress. In the current investigation, sampling-location parameters such as soil type, soil mound pH or chemical composition, precipitation, and temperature were uniform and corresponded with a total evenness value of 0.81. Even though these environmental parameters varied among the 3 sampling dates, no apparent trends could be noted. Community coefficient (CC) values across locations were similar between the corresponding substrates (e.g., plant debris–mound soil) and sampling dates, and the CC values were almost always similar with few exceptions. Even though CC values showed greater overall bacteria taxa variations within location and substrates, there were no significant differences between them. Rainfall amounts for each area were monitored via the Western Region Climate Center (WRCC 2013). Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 94 Precipitation varied from January through May and June ranging from 50 cm to over 250 and 300 cm, respectively. The 300-cm total occurred 30 days prior to the July sampling but did not significantly impact the CC data. Starting in July, the rainfall totals decreased overall to an average of 65 cm per month and was generally uniform through the last sampling in November. However, contrary to heavier rainfall midseason, species richness was greatest during the first sampling date. Temperatures increased from January (9 ºC) through July (26 ºC) and decreased from August (23 ºC) through December (8 ºC) with no apparent affect on CC values or species richness. As stated above, diversities and densities of bacteria collected from RIFA came from similar soil types at each of the 3 locations sampled along the Natchez Trace Parkway. These sites consisted of fine-silty or a mixture of fine-silty and fine-loamy soils (Brass et al. 2009, Cole et al. 1979, Scott et al. 1984). The slight variation of soils could have potential impact on the occurrence of microorganisms, but further studies would be needed for confirmation. In addition, when the imported fire ants construct their mound, it involves the mixing of soil materials from different depths including topsoil and subsoil (Green et al. 1998, Pettry 1999). This mixing generally results in higher clay content and lower sand and/or silt, which could affect the microorganism composition at each location. Various bacterial identification methods were used in this study with a goal to sort the isolates into distinct taxonomic units or groups and to confirm the names for the large numbers of bacterial isolations. Fatty acid components were a reliable and repeatable tool for identifying morphological groupings of isolates, especially if multiple genotypic strains or isolates formed a species complex (e.g., Bacillus spp.). Even though the fatty acid library results were consistent with replicated testing of morphologically similar isolate groupings, taxonomic names varied at a high rate compared to the 16S molecular results. As stated previously, molecular data of 16S rDNA sequences are considered the most accurate for species determination (Forbes et al. 1998, Jones and Bej 1994). Final determinations are shown in Appendix 1. The authors in those previous studies stated that 16S sequence data sensitivity to subspecies is often limited. In this study, fatty acid data for the pooled isolates overall yielded different species epithets than using 16S data from the Genbank Library but the two gave similar results at the generic level. It is uncertain why 16S data did not correspond with FAME’s library names since the latter method is still considered a reliable bacterial identification tool. It is noted that the MIDI FAME’s library emphasizes more economically important bacteria of human, animal, and agricultural or plant pathogens than general environmental surveys of taxa associated with ant communities. Bacillus sp. (5) listed in Appendix 1 was identified using 16S and had the highest isolation frequencies of any species during the study; this species was earlier identified as Bacillus thuringiensis from the MIDI library. This bacterial species was identified using 16S sequence data from another morphological isolate group but frequencies were low. Bacillus thuringiensis, which is a soil bacterium, is used as a biological pesticide for insect pests of cotton and other field crops. The implications of Bacillus thuringiensis presence in fire ant mounds are unknown. The need for Southeastern Naturalist 95 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 further screening of this bacterial species is necessary to determine the exact role of this bacterium in the mound ecosystem of RIFA. In conclusion, a diverse bacterial community of 68 culturable taxa were isolated from RIFA mounds collected along Natchez Trace Parkway in Mississippi. Final confirmation of the taxa identities were based on 16S sequence data, but fatty acid profiles derived from MIDI FAME’s library were used for initial groupings of the taxa and were supported by species identification data obtained from the Biolog System. Species richness, diversity, and evenness differed between sampling dates, substrates, and locations, but we noted no apparent trends. Some of the bacterial taxa isolated in this study were previously reported to have biological control potential with other insect species. Stored cultures of bacterial species from the mounds have been maintained for traditional confirmation of their taxonomic names and for use in future biological control or ant/microbe interaction studies. Currently, biocontrol studies with the bacterial isolates collected in this study are being evaluated for their antagonism or parasitism to RIFA as part of an ongoing USDA/ARS funded project. Acknowledgments The authors would like to acknowledge USDA-ARS Specific Cooperative Agreement for providing the support for this study under Project Number 6402-22320-00300D and the National Institute of Food and Agriculture, US Department of Agriculture, under Project No. MIS-012040. We are grateful to the Sigma Xi for its Grants-In-Aid Research in 2004. Finally, the authors are grateful to Mississippi State University (MAFES publication number 12420) for providing field and laboratory research facilitie s and supplies. The research presented in this paper represents a portion of S. Woolfolk's Ph.D. from Mississippi State University. The first and last listed authors contributed equally to this pa per. Literature Cited Amann, R.W., R.W. Ludwig, and K-H. Schleifer. 1994. Identification of uncultured bacteria: A challenging task for molecular taxonomists. American Society Microbiology 60:360–365. Baird, R.E., S. Woolfolk, and C.E. Watson. 2007. Survey of bacterial and fungal associates of black/hybrid imported fire ants from mounds in Mississippi. Southeastern Naturalist 6:615–632. Barron, G.L. 1972. Genera of Hyphomycetes from Soil. Robert E. Krieger Publishing Co., Malabar, FL. 364 pp. Beckham, R.D., S.L. Bilimoria, and D.P. Bartell. 1982. 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Spira. 2002. Biodiversity of fungi in Red Imported Fire Ant (Hymenoptera: Formicidae) mounds. Annual Entomology Society America 95:487–491. Southeastern Naturalist 99 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 Appendix 1. Mean percent isolation frequencies of bacterial taxa identified from Solenopsis invicta mounds from 3 locations (Hinds, Madison, and Leake counties) along Natchez Trace Parkway in Mississippi. Total % by substrateB NCBI Ant tissue Ant tissue Mound Mound Accesion external internal soil plant debris Overall Taxa no.A (AE) (AI) (MS) (PLANT DEBRIS) total % Achromobacter xylosoxidans 1 (Yabuuchi & Yano) Yabuuchi and Yano KF624697 0.0 0.6 1.1 0.0 less than 1.0 Achromobacter xylosoxidans 2 KF624698 6.7 3.3 19.4 7.8 9.0 Acinetobacter guillouiae Nemec et al. KF624699 0.0 1.1 8.9 0.0 2.5 Alcaligenes faecalis Castellani & Chalmers KF624696 0.0 2.2 2.8 0.6 1.4 Bacillus anthracis Cohn KF624700 0.6 0.0 0.0 0.6 less than 1.0 Bacillus cereus 1 Frankland & Frankland KF624701 0.0 0.0 0.6 0.0 less than 1.0 Bacillus cereus 2 KF624702 2.2 1.7 15.0 6.7 6.4 Bacillus cereus 3 KF624703 1.1 0.6 8.9 2.8 3.3 Bacillus megaterium de Bary KF624704 0.6 0.6 11.7 0.0 3.2 Bacillus pseudomycoides Nakamura KF624705 11.7 3.9 0.6 0.6 4.2 Bacillus sp. 1 Cohn KF624706 0.0 0.0 0.0 0.6 less than 1.0 Bacillus sp. 2 KF624707 0.0 2.2 13.3 0.6 4.0 Bacillus sp. 3 KF624708 0.0 0.0 1.1 0.6 1.0 Bacillus sp. 4 KF624709 0.6 0.0 1.7 0.6 less than 1.0 Bacillus sp. 5 KF624710 30.6 1.1 61.1 26.1 29.4 Bacillus sp. 6 KF624711 1.7 4.4 1.7 1.7 2.4 Bacillus sp. 7 KF624712 0.6 1.7 0.0 0.0 less than 1.0 Bacillus sp. 8 KF624713 0.6 0.6 8.3 5.6 3.8 Bacillus subtilis (Ehrenberg) Cohn KF624714 0.0 0.0 1.1 0.6 less than 1.0 Bacillus thuringiensis Berliner KF624715 0.0 1.1 10.0 0.0 2.8 Brevibacterium frigoritolerans Delaporte & Sasson KF624740 0.0 0.6 2.2 0.0 less than 1.0 Brevibacillus laterosporus (Laubach) Shida et al. KF624716 0.6 1.1 20.0 0.0 5.4 Brevundimonas diminuta Leifson & Hugh KF624717 0.0 1.2 2.2 0.0 1.1 Burkholderia sp. Yabuuchi et al. KF624718 0.0 0.0 2.8 1.1 less than 1.0 Carnobacterium maltaromaticum (Miller et al.) Mora et al. KF624719 0.0 0.0 0.6 0.0 less than 1.0 Collimonas pratensis Höppener-Ogawa KF624720 0.6 0.0 0.6 1.1 less than 1.0 Delftia lacustris Jørgensen et al. KF624721 0.0 0.0 2.8 0.0 less than 1.0 Southeastern Naturalist S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 100 Total % by substrateB NCBI Ant tissue Ant tissue Mound Mound Accesion external internal soil plant debris Overall Taxa no.A (AE) (AI) (MS) (PLANT DEBRIS) total % Enterobacter amnigenus Izard et al. KF624722 2.8 7.2 1.1 0.0 2.6 Enterobacter ludwigii Hoffmann et al. KF624723 1.1 0.0 1.1 2.8 1.3 Enterobacter sp. 1 Hormaeche & Edwards KF624724 0.6 5.0 2.2 0.0 1.9 Enterobacter sp. 2 KF624725 0.6 0.0 0.0 0.6 less than 1.0 Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Balz KF624726 0.0 0.0 1.1 0.0 less than 1.0 Jeotgalicoccus halotolerans Yoon KF624727 0.0 2.2 0.6 0.0 less than 1.0 Klebsiella oxytoca (Flugge) Lautrop KF624728 2.2 0.6 2.8 0.0 1.4 Lysinibacillus boronitolerans Ahmed et al. KF624729 3.3 0.0 10.6 11.1 6.3 Lysinibacillus fusiformis 1 (Priest et al.) Ahmed et al. KF624730 15.6 3.9 0.0 2.2 5.0 Lysinibacillus fusiformis 2 (Priest et al.) Ahmed et al. KF624731 0.0 0.0 1.7 0.6 less than 1.0 Lysinibacillus sphaericus (Meyer & Neide) Ahmed et al. KF624732 2.2 0.0 15.0 7.2 6.1 Lysinibacillus sp. Ahmed et al. KF624733 0.6 0.0 1.7 1.1 less than 1.0 Paenibacillus barcinonensis Sánchez et al. KF624734 0.0 0.0 3.9 1.1 1.3 Paenibacillus lautus (Nakamura ) Heyndrickx et al. KF624738 1.1 0.6 1.1 0.6 less than 1.0 Paenibacillus macerans (Schardinger) Ash et al. KF624735 0.0 0.6 2.8 1.7 1.3 Paenibacillus popilliae (Dutky) Pettersson et al. KF624736 1.1 0.0 0.6 1.1 less than 1.0 Paenibacillus sp. 1 Ash et al. KF624737 0.6 0.6 0.6 0.6 v1.0 Paenibacillus sp. 2 Ash et al. KF624739 3.9 1.7 6.7 3.9 4.0 Paemobacillus motobuensis Iida et al. 2005 KF624741 0.0 0.0 1.7 0.0 v1.0 Paenibacillus alvei (Cheshire & Cheyne ) Ash et al. 1994 KF624742 0.6 0.0 2.8 0.6 less than 1.0 Paenibacillus sp. Ash et al. 1994 KF624743 0.6 1.1 10.0 1.7 3.3 Pandoraea sp. 1 Coenye et al. KF624744 0.0 0.6 2.2 0.0 less than 1.0 Pandoraea sp. 2 KF624745 0.0 1.1 0.0 0.0 less than 1.0 Pandoraea sp. 3 KF624746 0.0 0.6 0.0 0.0 less than 1.0 Pseudomonas protegens Ramette et al. 2012 KF624747 5.0 3.9 11.2 5.0 6.2 Pseudomonas putida (Trevisan) Migula KF624748 3.3 2.2 1.1 1.1 1.9 Pseudomonas sp. 1 Migula KF624749 8.9 5.0 1.7 5.0 5.4 Pseudomonas sp. 2 KF624750 7.2 2.8 8.3 5.6 5.8 Serratia liquefaciens (Grimes & Hennerty) Bascomb et al. 1971 KF624751 8.3 12.8 5.0 1.1 6.3 Serratia marcescens 1 Bizio KF624752 0.0 0.0 0.6 0.0 less than 1.0 Southeastern Naturalist 101 S. Woolfolk, C.E. Stokes, C. Watson, R. Brown, and R. Baird 2016 Vol. 15, No. 1 Total % by substrateB NCBI Ant tissue Ant tissue Mound Mound Accesion external internal soil plant debris Overall Taxa no.A (AE) (AI) (MS) (PLANT DEBRIS) total % Serratia marcescens 2 KF624753 2.8 1.7 3.3 1.1 2.2 Serratia marcescens 3 KF624754 5.0 4.4 0.6 1.1 2.8 Serratia sp, 1 Bizio KF624755 0.0 1.1 1.7 0.0 less than 1.0 Serratia sp. 2 KF624756 4.4 0.0 1.7 0.6 1.7 Serratia sp. 3 KF624757 2.2 2.2 3.9 0.6 2.1 Serratia sp.4 KF624758 2.2 3.9 4.4 1.1 2.9 Staphylococcus epidermidis (Winslow & Winslow ) Evans KF624759 0.0 1.1 1.1 0.6 less than 1.0 Stenotrophomonas maltophilia (Hugh) Palleroni & Bradbury KF624760 0.0 0.6 0.0 0.0 less than 1.0 Unknown Bacterium sp. 1 KF624761 0.6 3.9 7.2 1.1 3.2 Unknown Bacterium sp. 2 KF624762 0.0 0.0 0.6 0.0 less than 1.0 Unknown Bacterium sp. 3 KF624763 0.0 0.0 1.1 5.0 1.5 AThe National Center of Biotechnology Institute (NCBI) accession numbers listed are based on submitted sequences of bacterial isolates to GenBank database. BMean percent isolation from soil mounds and plant debris is based on the percent occurrences of bacterial species isolated from three sampling dates (March, July, and November 2004), three locations (Hinds, Madison, and Leake Counties)/sampling date, five active mounds/location/sampling date, four replicates/mound/location/sampling date: Mean percent ¸ 180 (= 5 mounds × 3 locations × 3 sampling dates × 4 replicates) × 100. Overall mean total percentages of total bacterial isolates = (total mean percent isolation from all substrates ¸ 4) x 100. For ant body tissues the formula includes external and internal tissue samples: Mean percent ¸ 180 (= 5 mounds × 3 locations × 3 sampling dates × 4 replicates × 2 ant tissue types) × 100.