Simple, Filter-based PCR Detection of Thelohania solenopsae (Microspora) in Fire Ants (Solenopsis invicta)

THE red imported fire ant (Solenopsis invicta) is a serious introduced pest that is found throughout the southern USA. One factor contributing to its rapid spread is a lack of natural enemies (Jouvenaz 1983; Porter et al. 1997). A microsporidian parasite, Thelohania solenopsae (Allen and Buren 1974), has been identified as a fire ant pathogen in South America. Members of the phylum Microspora are unicellular, obligate intracellular organisms that infect a variety of vertebrate and invertebrate hosts (Becnel and Andreadis 1999).

Ant colonies infected with T. solenopsae had less brood than control colonies, and infected queens produced fewer eggs and had decreased survival times eventually resulting in colony death (Williams, Oi, and Knue 1999). Therefore, these parasites deserve further investigation as possible biological control agents to replace or augment chemical pesticide control programs.

Thelohania solenopsae was recently discovered in populations of 5. invicta in the USA (Williams, Knue, and Becnel 1998). However, the geographic distribution and prevalence of infection of the parasite in fire ants or native ants in this country are unknown. Before the parasite can be exploited as a biological control agent, additional biological and epidemiologic data are essential. Therefore, rapid, sensitive tools, including a molecular method for identification, are needed to facilitate field studies and to compliment microscopic methods for detecting parasites in ants.

MATERIALS AND METHODS

Preparation of ants. Uninfected and Thelohania solenopsae-infected colonies of Solenopsis invicta were routinely available from S. B. Vinson (Department of Entomology, Texas A&M University). Groups of 50–300 mg of ants (approximately 100 to 600 worker ants) from four infected colonies and one uninfected colony were frozen and homogenized in Tris buffered saline (25 mM Tris, 150 mM NaCl, pH 7.4) (TBS) with a Tenbroeck glass tissue grinder. Large pieces were allowed to settle 1 min, and a 10-ml suspension was transferred to a 15-ml conical tube and centrifuged for 10 min at 1,500 g. The supernatant was discarded, and the pellet was washed twice by resuspending in TBS and centrifuging. After three washes, the pellet was used for DNA preparation. A sample of each homogenate was prepared for microscopic evaluation by Calcofluor white M2R staining (Didier et al. 1995) to confirm the presence of parasite spores. For selected studies, washed spores were resuspended in 1 ml TBS and quantitated on a hemacytometer.

DNA preparation. DNA template for PCR reactions was prepared from the homogenate using two different methods. In the first technique, DNA was extracted in a conventional system using a GenElute Mammalian Genomic DNA Kit (Sigma, St. Louis, MO) following the manufacturer's instructions.

For comparison, an extraction-free, filter-based template preparation method was adapted for entomologic needs using a commercially available filtration matrix and washing buffer (FTA Classic Card and FTA Purification Reagent, Whatman BioScience, Newton, MA). This system consists of a membrane card that captures DNA and RNA for direct processing in a PCR reaction. Twenty microliters of the ant/parasite homogenate were applied to the FTA membrane surface, allowed to air dry and stored at room temperature. Two-millimeter samples were punched from the FTA card (Harris 2.00-mrn micropunch, Whatman BioScience, Newton, MA), placed in 1.5-ml microtubes, and washed with the FTA Purification Reagent and with TE buffer (10 mM Tris +1 mM EDTA, pH 8.0, Sigma, St Louis, MO) following the manufacturer's instructions.

Alternative ant preparation. One concern with DNA preparation from ants is the reuse of Tenbroeck tissue grinders which could result in DNA cross-contamination. As an alternative preparation method, ants were processed using a bead-beating system with disposable microtubes. Fifty to 300 mg of ants were added to a microtube with 750 mg of 1.0-mm glass beads in 1 ml of TBS and homogenized in a mini-bead beater (Mini-beadbeater Type BX-4 cell disrupter, Biospec Products, Bartlesville, OK) for 15 sec at 5,000 rpm. Homogenate was subsequently applied to the FTA Card, dried, and processed as previously described.

Molecular detection of T. solenopsae from naturally infected ants. A portion of the small subunit ribosomal RNA gene (ssu rRNA) was selected as the diagnostic target for the parasite. Several primer pairs were designed based on alignments of published ssu rRNA gene sequences from Thelohania (Genbank Accession # AF134205, AF031538 and AF031537) using MacVector software (MacVector 7.0, Oxford Molecular Ltd., Madison, WI). One primer pair that amplified a 366-BP section of the ssu rRNA gene included the Thelohania-specific forward primer, Th698F (5′-GATGATTAGATACCGTTGTAGTTCC- 3′), and the reverse primer, Th1064R (5′-GCTTACCGCAAGAAGTCTCAC- 3′), which amplifies several microsporidia species. A second primer pair that amplified a 1073-BP portion of the gene included the Thelohania-specific forward primer, Th172F (5′-GAAAGCGGAGCATCATTGTAGG- 3′), and the reverse primer, Th1245R (5’-TTCATCGTTACTTAG- TGAGCAGCG- 3′), which is specific for Thelohania but shares close homologies with several microsporidial species.

All PCR reactions were performed in 25-μl reaction-mixture volumes using reagents and manufacturer's instructions for the Taq polymerase (JumpStart RED Accutaq DNA Polymerase, Sigma, St. Louis, MO) with the addition of acetylated BSA at 0.1 μg/μl. Amplifications were performed using a stepdown program (24 cycles using 94°C for 1 min; stepdown 65°C to 47°C in 3 degree steps for 2 min; 68°C for 3 min; followed by 15 cycles using 94°C for 1 min; 44°C for 2 min; 68°C for 3 min).

Titration of parasites to determine assay sensitivity. In order to estimate the sensitivity of each DNA preparation method, washed spores were quantitated with a hemacytometer. Using the GenElute extraction kit (Sigma, St. Louis, MO), quantified spores were added in 10-fold dilutions (1 × 10⁶ to 1 × 10¹ spores) in the kit's resuspension solution and individually extracted.

To accurately prepare FTA membrane punches with defined quantities of spores, 2.00-mm membrane discs were placed in 1.5-ml microtubes. Spores of eight concentrations (1 × 10⁴ to 1×1¹ pores) were applied in 1 μl volumes directly to the discs, dried and processed following manufacturer's instructions. The primer pair Th698F and Th1064R was used to compare detection sensitivity between the two DNA preparation methods. PCR amplification and detection of DNA prepared by both methods were conducted as described above.

RESULTS AND DISCUSSION

A simple, extraction-free method was developed to detect T. solenopsae parasites in naturally infected fire ants. Using a conventional DNA extraction method and using the FTA Classic Card system, parasites were detected in groups of ants from four infected colonies using PCR amplification of a portion of the ssu rRNA gene. In six experiments comparing DNA from the two preparation methods, appropriately sized amplicons were produced in all reactions ensuring that the FTA system worked as effectively as conventional DNA extraction methods. As a control, ants from an uninfected colony produced no PCR products in three separate experiments. To ensure repeatability, three different groups of ants from one infected colony were processed on two separate occasions (six replicates), and appropriately sized amplicons were consistently produced.

Additionally, a titration of quantitated spores was used to compare the detection sensitivities of the conventionally processed DNA and the FTA card system. The GenElute extraction kit produced consistent amplification with spore concentrations as low as 1 × 10³ organisms. In comparison, the FTA membrane was more sensitive, consistently detecting 1 × 10² spores. This finding is similar to the sensitivity level included in a recent report that used FTA Card technology to detect several enteric parasites in human diarrheic samples (Orlandi and Lampel 2000).

The FTA Classic Card technology is used in an expanding range of applications, such as genetic studies with human blood and tissues (Devost and Choy 2000), detection of viral, bacterial, and parasitic pathogens (Beck et al. 2001; Lampel, Orlandi, and Kornegay 2000; Orlandi and Lampel 2000), and detection of plant genes (Natarajan et al. 2000). Advantages associated with this entomological study are that the FTA card containing the ant materials can be stored at room temperature and provides material for repeated processing or future DNA retrieval. Using the FTA card system also eliminates the time-consuming DNA extraction process, which facilities the rapid processing of a large number of samples in field surveys.

Comparisons of ant preparations completed with the Tenbroeck tissue grinder and with bead beating showed equal success in subsequent detection of T. solenopsae. The bead-beating method eliminated the possibility of cross-contamination when compared to the tissue grinder since the bead-beating microtubes are disposable. Therefore we recommend preparation by bead-beating prior to use of the FTA Classic Card System.

The FTA card method can be used in conjunction with microscopy to identify infected fire ant colonies. Molecular detection of parasites has an advantage over microscopy because the microsporidia can be identified to the genus or species level, which is not possible using light microscopy. While parasites are reportedly easy to detect through individual ant dissection (Knell, Allen, and Hazard 1977), numerous ants may need to be processed if the parasite infection rates are low in ant colonies. The FTA card system can be used to evaluate groups of ants with a sensitive threshold of parasite detection, thereby eliminating tedious microdissection of large numbers of ants.

In summary, the FTA format is a useful tool which provides a simple, rapid, extraction-free method for the molecular detection of the microsporidian parasite, T. solenopsae in the fire ant, S. invicta. This report adds to the growing number of applications for the FTA filter methodology, which has been adapted to a wide variety of clinical, food, and environmental sources for molecular studies and archival storage of DNA samples.