Sample collection of Reticulitermes speratus and Reticulitermes kanmonensis

To design a diagnostic method based on species-specific genetic sequences for R. kanmonensis, the native species R. speratus was collected and used as a control for comparative analysis. Reticulitermes speratus was collected in June and August 2023 from two locations: Wanju-gun in Jeollabuk-do, where R. kanmonensis was first identified in Seocheon-gun in Chungcheongnam-do, South Korea (Lee et al., 2015). R. speratus samples were sourced from both standing and fallen dead pine trees or stumps using an axe; R. speratus specimens collected from a single stump and tree were treated as a single sample and preserved in 95% ethanol. Four samples, each comprising a minimum of one army ant and two worker ants, were collected and utilized (Fig. 1).

Sequencing

The Clear-S Quick DNA extraction kit (IVT-3002, Invirustech, S. Korea) was used to extract DNA from the collected termite samples according to the manufacturer’s protocol.

Production of primers for gene amplification and selection

To design primers for species amplification, this study identified candidate sequences from WGS analysis of genomic DNA. Subsequently, Primer3 software (Untergasser et al., 2012) was employed to design five pairs of primer and probe sets for each species (Table 1), focusing on the precise amplification of species-specific genetic sequences. The properties of these primers were optimized considering Tm values, GC content, and potential secondary structure formation.

qPCR was conducted using extracted and refined genes from two species as templates. The conditions were as follows: 10 μL EzAmp^TM HS qPCR 2X Master Mix (SYBR Green, Low Rox), 3 μL (1 ng/μL) of genomic DNA, 0.5 μL of each primer at 10 pmol, and 6 μL of nuclease-free water for a final volume of 20 μL. The cycling condition commenced with an initial denaturation at 95°C for five minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60°C for 30 seconds. Melting curve analysis was performed, increasing the temperature by 0.5°C/s from 60°C to 95°C. Two primer sets were selected for each species based on their PCR amplification performance and specificity.

Whole-genome sequencing (WGS)

WGS was conducted using the Illumina Novaseq platform with 150 bp libraries in a paired-end sequencing format, generating each 150 bp read. This sequencing produced 282,273,060 RAW reads with Q20 (97.09%), Q30 (92.39%), and GC content (46%). WGS data for other termite species were obtained from the SRA database of NCBI GenBank. WGS data estimated the total genome size of R. kanmonensis to be approximately 270-277 Mb, of which about 81% was repetitive sequences.

For the effectiveness of the designed primers, it is crucial that they exhibit a low mutation rate. HMMER and BLASTn analyses of the designed primers and amplified sequences revealed that all five sets showed high similarity to protein-coding regions of other termites, suggesting these as potential protein-coding regions of R. kanmonensis (Tables 2 and 3). Nevertheless, some primer sets did not align with specific protein-coding regions, indicating the need for further research, including a full genome analysis of R. kanmonensis.

Development and selection of species-specific primers

A TaqMan-based probe method, known for its high specificity and sensitivity, was employed to enhance accuracy. DNA was extracted from each termite, followed by WGS to identify species-specific sequences. Subsequently, PCR results were confirmed, and target sequences were found to be well-amplified (Table 4). By selecting and verifying primer sets that efficiently amplify only the target species, R. Kan set 1 and R. Kan set 4 were chosen as species-specific primers for R. kanmonensis due to their high amplification efficiency and lack of non-specific bands (Fig. 2).

Specificity and sensitivity of species-specific primers

In qPCR, the Ct value, representing the Cycle Threshold, is used to estimate the initial amount of target DNA. Given the higher amplification sensitivity, even a low initial concentration of target DNA can be detected, resulting in a lower Ct value. To confirm the sensitivity of species-specific primers, the Ct value of each primer set was determined. The primer set with the lowest Ct value was selected as the species-specific marker.

Although the initially selected primer set was adequate to obtain results, the Ct values were compared to ensure more reliable and accurate outcomes (Fig. 3). In the case of R. kanmonensis, the Ct value for R. Kan set 1 was 30.945, indicating higher sensitivity compared to R. Kan set 4 (Table 5). Further analysis of PCR gel loading results (Fig. 2) showed that in R. kanmonensis, R. Kan set 1 amplified more specifically than R. Kan set 4, suggesting that R. Kan set 1 is likely more effective.