The Formosan subterranean termite (FST) Coptotermes formosanus Shriaki (Isoptera: Rhinotermitidae), is a major economic pest worldwide and has become a serious pest to the United States and its territories. Native to China, FST has been introduced into Japan, Guam, Sri Lanka, South Africa, Hawaii and the continental United States (Mori 1987; Su & Tamashiro 1987; Wang & Grace 1999). Coptotermes formosanus was first recorded in continental United States at Charleston, SC in 1957 (Chambers et al. 1988). Numerous well-established colonies were discovered in Florida in 1980, 1982, and 1984 (Oi et al. 1992) with many additional finds since (Su & Scheffrahn 2000). Introductions to San Diego, CA (Atkinson et al. 1993; Haagsma et al. 1995), the Gulf Coast states, and southeastern US also have been documented (Spink 1967; LaFage 1987; Su & Tamashiro 1987; Howell et al. 1987; Appel & Sponsler 1989; Oi et al. 1992; Su & Scheffrahn 1998; Hawthorne et al. 2000; Howell et al. 2000; Scheffrahn et al. 2001; Hu et al. 2001; Jenkins et al. 2002). Since 2002, C. formosanus has been considered a quarantine pest in Mississippi (Mississippi Department of Agriculture and Commerce, Rule 40). In the city of New Orleans, the control and repair costs due to FST are estimated at $300 million annually (Lax & Osbrink 2003) and annual damage to the entire United States is estimated to exceed $1 billion. It is considered the single most economically important insect pest in the state of Hawaii (Su & Tamashiro 1987).

The inability to quickly discriminate what Coptotermes species one is dealing with could lead to difficulties in evaluating the source of the infestation. While introductions of FST have gained recent notoriety, less is reported or known about other potentially damaging Coptotermesspecies. Exotic introductions of Coptotermes havilandi to Florida (Su et al. 1997; Su et al. 2000; Scheffrahn & Su 2000) have been detected. Although not established, C. havilandiHolmgren, which recently has been synonymized as C. gestroi (Wasmann) by Kirton & Brown (2003), has been recovered in shipping crates in Tennessee imported from East Asia (RHS, unpublished data). Coptotermes from South America, Central America, and the Caribbean also pose potential problems for the US. There are three described endemic species of Coptotermes in the Americas including C. crassusSnyder, C. testaceus L., and C. niger Snyder, and possibly others which have not been identified. More recent genetic surveys have uncovered old world Coptotermes species, C. sojesti, introduced to the West Indies (Scheffrahn et al. in press).

Mistakes have been made in both the naming and correct identification of some Coptotermes. For example, the inconsistencies in the pest status of C. havilandi in different regions of its geographic range have been due to misidentification and taxonomic confusion between C. travians(Haviland), C. havilandi, and C. gestroi (Kirton & Brown 2003). An examination of the type series of C. travians indicates the species has been misidentified in Peninsular Malaysian literature (Tho 1992) as C. havilandi and also has been referred to as C. borneensis Oshima (Kirton & Brown 2003).

Correct identification is critical for pest insects. Identification of termite workers is possible at the generic level only, and finding an alate, which can be identified, in a collection is seasonal and can be rare. We have developed a molecular diagnostic method capable of differentiating FST from other Coptotermes species regardless of the caste encountered or locality obtained. It has been demonstrated that both nucleotide sequencing and restriction enzyme digestion of polymerase chain reaction (PCR) amplicons can be used to differentiate between various termite species (Austin et al. 2002; Austin et al. 2004; Szalanski et al. 2003; Clement et al. 2001; Jenkins et al. 2002; Uva et al. 2004). DNA sequence differences reported between Coptotermes species from numerous disjunctive populations from around the world in a small portion of the mtDNA were exploited to design species-specific PCR primers and to develop a DNA-based assay that can discriminate FST from other Coptotermes species.

Materials and Methods

Coptotermes termites were collected from various locations in North America, South America, the Caribbean, Australia, Africa, and Asia (Table 1). Morphological identification of specimens used in this study was achieved by using Snyder (1922), Emerson (1925), Emerson (1928), Hill (1942), Scheffrahn et al. (1990), Scheffrahn & Su (1994), Tho (1992), and Su et al. (1997). Voucher specimens, preserved in 100% ethanol, are maintained at the Arthropod Museum, Department of Entomology, University of Arkansas, Fayetteville, AR, USA.

Alcohol preserved specimens were allowed to dry on filter paper, and DNA was extracted from individual worker or soldier heads with the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis, MN). Extracted DNA was resuspended in 50 μl of Tris:EDTA and stored at -20°C. Polymerase chain reaction was conducted with the primers LR-J-13007 (5’-TTACGCTGTTATCCCTAA-3’) (Kambhampati & Smith 1995) and LR-N-13398 (5’-CGCCTGTTTATCAAAAACAT-3’) (Simon et al., 1994). These PCR primers amplify an approximately 428-bp region of the mtDNA 16S rRNA gene. PCR reactions were conducted with 1 μl of the extracted DNA per Szalanski et al. (2000), and a profile consisting of 35 cycles of 94°C for 45 s, 46°C for 45 s and 72°C for 45 s. Amplified DNA from individual termites was purified and concentrated by Microcon-PCR Filter Units (Millipore, Bedford, MA). Samples were sent to The University of Arkansas Medical School DNA Sequencing Facility (Little Rock, AR) for direct sequencing in both directions with an ABI Prism 377 DNA sequencer (Foster City, CA). GenBank accession numbers for the Coptotermes termites subjected to DNA sequencing in this study are AY558898 to AY558914. DNA sequences were aligned by the PILEUP command of GCG (Accelrys, San Diego, CA), and the distance matrix option of PAUP* 4.0b10 (Swofford 2001) was used to calculate genetic diversity according to the Kimura 2-parameter model (Kimura 1980) of sequence evolution.

Results and Discussion

The 428-bp region of the mtDNA 16S rRNA gene was subjected to DNA sequencing from 12 described species of Coptotermes, including 64 populations of C. formosanus (Table 1). Within the genus, genetic diversity ranged from 1.8% between C. testaceus and C. crassus to 7.0% between C. acinaciformis and C. vastator. No genetic variation was observed between the two C. testaceus samples, and up to 0.7% variation was observed among the C. gestroi samples. No genetic variation was observed in C. formosanus, and C. formosanus was most similar to the C. intermedius sample from Togo Africa, with 3.0% DNA sequence divergence. Phylogenetically, C. formosanus forms a distinct clade among non-Australian Coptotermes (C. vastator, C. testaceus, C. crassus, C. sjostedti, C. intermedius, C. gestroi, C. heimi, and C. carvinatus) (Scheffrahn et al. in press).

Formosan subterranean termite 16S DNA sequences along with sequences from other Coptotermes, Reticulitermes (Szalanski et al. 2003) and Heterotermes (Szalanski et al. 2004) were aligned and examined for mismatches that reflected either substitutions or deletions. The mismatches were exploited to design primers that were unique to FST (Table 2). Two primers, one from each strand FST-F (5’-TAAAACAAACAAACAACAAACAAAC-3’) and FST-R (5’-ATGGCTTGACGAGGCACAA-3’) were designed. Based on the sequence, the expected sizes of the amplicon is 151 bp. Proper sized PCR products were obtained with conspecific DNA, whereas no product was obtained with template from the other species, i.e., no false positives were observed with known DNA. Each FST primer paired with a common primer will only amplify Formosan subterranean termite DNA.

The FST species-specific primers were tested for optimal annealing performance in a 47°-59°C temperature gradient with 2°C intervals. The optimal annealing temperature for the FST specific primers was 57°C. PCR reactions were the same as the 16S conditions with the exception of the PCR profile which consists of 30 cycles of 94°C for 45 s, 57°C for 45 s and 72°C for 45 s. This annealing temperature, however, is too high for the 16S universal primers, preventing multiplex PCR with both primer pairs. To resolve this, both PCR reactions are conducted individually and 10 μl of each PCR reaction are loaded onto a single well of a 2% agarose gel (Fig. 1). The FST specific primer set successfully amplified for 52 individual FST from all 23 FST populations, and did not yield a PCR product for the other Coptotermes, Reticulitermes and Heterotermes. PCR product from samples that are not C. formosanus can be subjected to DNA sequencing and compared with the database of termite 16S sequences on GenBank for identification by a BLAST search ( http://www.ncbi.nlm.nih.gov/BLAST/). This technique provides a method to identify field collected termites and facilitates the screening of the monitoring for this species and for the introduction of other invasive Coptotermes termites.

In the context of determining the species for a large number of samples collected in connection with distribution or competition studies, simplifying the identification of the worker caste is advantageous. FST has no genetic polymorphism across its geographic distribution for the 16S marker, whereas genetic variation has been observed in the mtDNA COII gene (ALS unpublished data, Jenkins et al. 2002). This lack of mtDNA 16S intraspecific variation makes this marker ideal for molecular diagnostics.

These primers provide a convenient way to identify individual termites without resorting to more time consuming restriction fragment-length polymorphism analysis, or extensive morphological data which may result in overlap due to clinal variations in size as observed in many insects including termites. The approach is equally applicable to other castes, such as soldiers and alates, but given their obvious taxonomic importance should be constrained to either morphologically ambiguous samples, or when the more diagnostic castes are unavailable. This should be an important new tool for substantiating the identity of FST before the onset of regulatory procedures (i.e., quarantine).