Description of a New Cryptic Rhabditid, Parasitorhabditis paraterebrana n. sp. (Nematoda: Rhabditidae), with Remarks on Two Known Species from Korea

Samples of the bark of dead pine-tree stands were taken from pine-tree forests in Uljin, and Gumi, Gyeongsangbuk-do Province, Republic of Korea. The nematode populations were extracted from the bark layer cuttings using the Baermann funnel method (Baermann, 1917). Nematode specimens belonging to Parasitorhabditis were handpicked from the collected nematode suspension under a Nikon SMZ1000 stereomicroscope (Nikon). Specimens were subsequently characterized based on inferences from morphometric and molecular data.

The specimens were heat-killed, fixed with formaline-glycerine, and transferred to pure glycerin according to Seinhorst (1959) as modified by De Grisse (1969). The processed nematode specimens were mounted on permanent slides and observed under a light microscope. Morphometric data and photomicrographs were taken using a Zeiss imager Z2 microscope (Carl Zeiss) fitted with Axiovision, Material Science Software for Research and Engineering (Carl Zeiss). Line drawings were made using a drawing tube and redrawn using CorelDRAW® software version 24. Species identification and diagnosis were done following the diagnostic species compendia presented by Rühm (1956), Andrássy (1983), and Carta et al. (2010).

Morphometrically confirmed, heat-relaxed female and male specimens were used in DNA extraction. Genomic DNA was extracted from the specimens using the method of Iwahori et al. (2000), with modifications. Briefly, nematodes were washed in drops of distilled water on a glass slide. A single cleaned nematode specimen was transferred into a 1-μl drop of distilled water on a sterilized glass slide and crushed with a small filter paper chip (1 mm × 1 mm) with the aid of a forceps under a stereoscope microscope. The paper chip was subsequently suspended in a 30 μl nematode DNA lysis buffer containing proteinase K and incubated at 65 °C for 30 mins and 95 °C for 10 mins. The resultant solution was used as the DNA template for a polymerase chain reaction (PCR). PCR was performed using WizPure™ Taq DNA Polymerase kit in accordance with the manufacturer’s instructions. Four gene fragments (the nearly full-length 18S rRNA gene, the D2-D3 expansion segment of 28S rRNA gene, the partial ITS rRNA gene and the partial COI gene) were amplified and sequenced in this study. The 18S rRNA gene was amplified as two partially overlapping fragments using two primer sets: 988F (5′-CTCAAAGATTAAGCCATGC-3′) and 1912R (5′-TTTACGGTCAGAACTAGGG-3′), 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and 2646R (5′-GCTACCTTGTTACGACTTTT-3′) (Holterman et al., 2006); the primer pairs D2Ab (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De ley et al., 1999) were used to amplify the D2-D3 expansion segment of 28S rRNA; COI-F1 (5′-CCTACTATGATTGGTGGTTTTGGTAATTG-3′) and COI-R2 (5′-GTAGCAGCAGTAAAATAAGCACG-3′) (Kanzaki and Futai, 2002) were used to amplify the partial COI gene; TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGG GT-3′) (Curran et al., 1994) were used to amplify the partial ITS rRNA gene of P. terebrana and P. paraterebrana n. sp.; and S_ITS1 (5′-TTGATTACGTCCCTGCCCTTT-3′) and Vrain2R (5′-TTTCACTCGCCGTTACTAAGGGAATC-3′) (Vrain et al., 1992) were used to amplify the partial ITS rRNA gene of P. obtusa. PCR was executed with a thermal cycler model T100™, Bio-Rad. The thermal cycling program using the primer sets 988F/1912R, 1813F/2646R, D2Ab/D3B, TW81/AB28, and COI-F1/COI-R2 were as described by Mwamula et al. (2023), and the thermal profile using S_ITS1/Vrain2R primer set was as detailed by Mwamula et al. (2024). Purification of the PCR products was performed using the QIAquick PCR Purification Kit (Qiagen) and quantification was done using a quickdrop spectrophotometer (Molecular Devices). The purified PCR products were directly sequenced with the primers specified above at Macrogen Inc. The edited new sequences were submitted to the NCBI GenBank database under the accession numbers: PQ589232-PQ589236 (for 18S rRNA); PQ589220-PQ589225 (for 28S rRNA); PQ589226-PQ589231 (for ITS rRNA), and PQ585733-PQ585737 (for the COI gene).

The new sequences (18S rRNA, D2-D3, ITS rRNA and COI gene) were compared with those of related species of Parasitorhabditis using the BLAST homology search program. Multiple alignments for sequences of the genus, including comparable sequences of related species from other genera published in GenBank (Kanzaki et al., 2013, 2015, 2016; Valizadeh et al., 2017; Bhat et al., 2020; Mwamula et al., 2023) were built using ClustalX (Thompson et al., 1997). The sequences of Rhabditoides inermiformis (Osche, 1952) Dougherty, 1955 (AF083017) and Rhomborhabditis regina (Schulte and Poinar, 1991) Sudhaus, 2011 (AF082997) were selected as the outgroup sequences for the 18S rRNA gene; sequences of Rhabditoides inermiformis (EF990727) and Rhomborhabditis regina (EF990726) were selected as outgroups for 28S rRNA gene; and those of Chylorhabditis epuraeae Kanzaki, Hamaguchi, and Takeuchi-Kaneko, 2021 (LC601016) and Rhabditis Dujardin, 1844 species (OL468734) were the outgroups for COI gene. The ITS rRNA gene phylogeny was not reconstructed. This is because, except for only four sequences of P. terebrana, there were no other definitive ITS rRNA gene sequences of Parasitorhabditis or other related genera in GenBank database for comparison. Bayesian inference (BI) of the three genes was performed using MrBayes 3.2.7 (Ronquist et al., 2012), with GTR + I + G model selected for all datasets. Bayesian analysis for each gene was initiated with a random starting tree and run with four chains for 1 × 10⁶ generations. The Markov chains were sampled at intervals of 100 generations. After discarding burn-in samples, consensus trees were generated with the 50% majority rule. The generated trees were visualized and edited using FigTree v1.4.4 software. Posterior probabilities (PP) exceeding 50% are given on appropriate clades. Intraspecific and interspecific sequence variations were analyzed using PAUP* v4.0a169 (Swofford, 2003).

See Table 1.

Parasitorhabditis paraterebrana n. sp. is characterized by having a medium sized body 0.55–0.81 mm long in both sexes; stoma 20.0–24.0 μm in depth; anterior tips of prorhabdions not bent inwards; remnants of metarhabdions with two subventral teeth, and two subdorsal teeth; corpus longer than postcorpus; hemizonid visible, 15.0–26.5 μm posterior to excretory pore; vulva-anus distance of 21.5–31.5 μm, ca equal to or slightly less than vulval body diameter, also ca equal or less than tail length; rectum distinctly longer than anal body diameter; female tail cupola-shaped, conoid posteriorly, with an extended moderate spike; spicules slender, nearly straight to minimally curved towards a nearly acute to a bluntly rounded tip; and bursa with 10 pairs of bursal rays with a 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3) typical ray pattern, occasionally appearing in a 2 + 4 + (1+3) pattern.

Parasitorhabditis paraterebrana n. sp. looks very similar to P. terebrana in morphology, having two visible subventral teeth, and two subdorsal teeth and sharing almost all morphometric values. However, it differs from P. terebrana by having non-bent anterior tips of prorhabdions vs. anterior tips of prorhabdions bent inwards (see Massey (1974) and Mwamula et al. (2023)), a corpus being longer than the postcorpus vs. being equal to or slightly shorter than the postcorpus; the rectum being distinctly longer than anal body diameter vs. being almost equal; bursal rays in the pattern 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3), occasionally appearing as 2 + 4 + (1+3) vs. 2 + (3+1) + 4; a more cupola-shaped tail in females; and differences in their DNA barcodes (details provided below). Based on morphometrics, Parasitorhabditis paraterebrana n. sp. is also close to P. crypturgophila Rühm, 1956, P. opaci Rühm, 1956, P. palliati (Fuchs, 1937) Skrjabin, Shikhobalova, Sobolev, Paramonov, and Sudarikov, 1954, P. ateri (Fuchs, 1915) Dougherty, 1955, P. autographi (Fuchs, 1937) Skrjabin, Shikhobalova, Sobolev, Paramonov, and Sudarikov, 1954 and P. frontali Carta, Bauchan, Hsu, and Yuceer, 2010. It differs from the P. crypturgophila by its longer stoma (20–24 vs. 16–19 μm), the presence of four teeth vs. no teeth, a corpus longer than the postcorpus vs. equal in length to postcorpus, longer gubernaculum (14–17 vs. 9–14 μm), and shorter vulva to anus distance (21.5–31.5 vs. 32–35 μm); from P. opaci by the longer stoma (20–24 vs. 17–19 μm), the presence of four teeth vs. three teeth, a cupola-shaped tail with an extended spike vs. conical tail; a lower b ratio (3.2–4.3 vs. 4.4–5.2); a shorter vulva to anus distance (21.5–31.5 vs. 30–35 μm), and bursal rays in the pattern 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3), occasionally appearing as 2 + 4 + (1+3) vs. 2 + 3 + 5. The new species differs from P. palliati by its shorter spicules (30.5–37.0 vs. 39–44 μm), a shorter gubernaculum (14–17 vs. 18–23 μm), bursal rays in the pattern 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3), occasionally appearing as 2 + 4 + (1+3) vs. 2 + 4 + 4, the presence of four teeth vs. three teeth, a shorter vulva to anus distance (21.5–31.5 vs. 32–35 μm), a lower b ratio (3.2–4.3 vs. 5–6), and a cupola-shaped tail with an extended spike vs. conical tail. The new species differs from P. ateri by having shorter spicules (30.5–37.0 vs. 39–42 μm), a shorter gubernaculum (14–17 vs. 18–21 μm), the presence of four teeth vs. two-three teeth, bursal rays in the pattern 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3), occasionally appearing as 2 + 4 + (1+3) vs. 2 + 4 + 4, a shorter vulva to anus distance (21.5–31.5 vs. 32–35 μm), a lower b ratio (3.2–4.3 vs. 4.9–6.6), and a cupola-shaped tail with an extended spike vs. conical tail. The new species differs from P. autographi by the presence of four vs. two teeth, lower b and c ratios (3.2–4.3 vs. 4.8–5.3 and 18.3–28.6 vs. 29–59, respectively), a shorter vulva to anus distance (21.5–31.5 vs. 35 μm), a longer pharynx (167–190 vs. 155–161 μm), the position of the vulva (V = 90.3–92.6 vs. 92–95), a cupola-shaped tail with an extended spike vs. a conical tail; and bursal rays in the pattern a 2 + 3 + 2 + 3 or 2 + 3 + (2 + 3), occasionally appearing as 2 + 4 + (1+3) vs. 2 + 5 + 3. The new species differs from P. frontali by the presence of four teeth vs. one tooth, a corpus longer than postcorpus vs. shorter than postcorpus, a longer pharynx (167–190 vs. 113–136 μm), a longer stoma (20–24 vs. 13–17 μm); and a longer gubernaculum (14–17 vs. 11.1–12.8 μm).

The species was recovered from the bark of a dead red pine (Pinus densiflora for. erecta) tree from Geumgang pine-tree forest in Uljin, Gyeongsangbuk-do Province. GPS coordinates: 37°02′84″N, 129°27′51″E.

The holotype female, twelve female paratypes, and fifteen male paratypes were deposited in the National Institute of Biological Resources of Korea and eight female and seven male paratypes were deposited in the Nematode Collection of Kyungpook National University (KNU), Republic of Korea.

The species epithet refers to the Greek preposition para, “alongside of,” resembling the morphologically closest species, Parasitorhabditis terebrana.

Korean population of Parasitorhabditis terebrana

See Table 1.

The morphological and morphometric data of the studied population agree well with those of the paratype specimens studied by Massey (1974), and the subsequent details given by Mwamula et al. (2023), except for the relatively long body in the latter population (780.5–995 vs. 922–1244 μm). Also, nine bursal rays were recorded in the original description by Massey (1974). However, 10 bursal rays are evident in the currently studied population, with the ray pattern similar to the descriptions given by Mwamula et al. (2023). This observation is also in agreement with Andrássy (1983) and Sudhaus and Fitch (2001), who noted that the number of bursal rays (papillae) is constant in the genus Parasitorhabditis, i.e., 10 pairs (two of the ten pairs preanal). According to Massey (1974), the diagnostic characters of the species include the corpus being equal in length to isthmus and basal bulb, the unique tip of the prorhabdions bent inwards and the tail terminus shape. These features are seen in the population recovered and studied here, and the DNA sequences are identical to the previously published sequences for P. terebrana (Mwamula et al., 2023). The reproductive system is generally similar to that of Parasitorhabditis paraterebrana n. sp., arranged from anterior (distal) as ovary, oviduct, spermathecal-uterus junction, crustaformeria, uterus, vagina/vulva, extending 46–57% of total body length. Distal part of ovary reflexed dorsally, covering 37–70% of the total gonad system length. Oocytes arranged in two to three rows in the reflexed part of ovary, and in a single row near oviduct. Oviduct comprised by small ellipsoidal cells, connected to an enlarged wider posterior part composed of large cells, forming a roundish to oblong-shaped sac. Spermathecal-uterus junction distinct, connected by a band of thread-like tissue. Uterus composed of irregularly arranged globular uterine cells, forming prominent crustaformeria, and the wider proximal tubular part with epithelial wall. The uterus often contains 1–3 developing eggs. Vagina short, oblique with muscular walls. Lips of vulva mostly protuberate. Vulva a transverse slit. Male with a single testis, on right subventral region of intestine, outstretched, extending 44–56% of total body length, anteriorly reflexed ventrally or dorsally up to 22–54% of its length. Spermatocytes arranged in two to three rows in reflexed part. Vas deferens tube-like, occupying ca 24–27% of total gonad length, composed of large cells, joining the rectum and forming a narrow cloacal tube terminating into the cloaca.

Korean population of Parasitorhabditis obtusa (Fig. 4)

Figure 4:

Photomicrographs of Parasitorhabditis obtusa (Fuchs, 1915) Chitwood and Chitwood, 1950 (A-H). A, B: Female anterior region; C, D: Male tail region; E, F, H: Variation in shape of female tail; G: Posterior part of pharynx (arrow e and h indicate the position of secretory-excretory pore and hemizonid, respectively). (A, B, E, F, H = 10 μm; C, D, G = 20 μm).

See Table 2.

The morphology and morphometrics of the studied population agree well with those of the type population described by Fuchs (1915) and the subsequent morphometric data published by Rühm (1956), Massey (1956, 1960), Carta et al. (2010), and Valizadeh et al. (2017) except for the number of bursal rays reported by Massey (1960), with only 9 bursal rays illustrated in the line drawings, and Valizadeh et al. (2017), who reported 8–9 pairs of bursal rays although 10 pairs were illustrated in their line drawings (in lateral view). Additionally, the bursal rays arrangement pattern is not definitive in Valizadeh et al. (2017) (i.e., 4 rays in precloacal position and 6 rays post-cloacal). In the population studied here, a pattern of bursal rays as 2 + 3 + 2 + 3 was consistent and rarely appeared in a 2 + 3 + 3 + 2 pattern, agreeing with the illustrations of Rühm (1956). Additional characters reported in this study include corpus equal to postcorpus, excretory pore located posterior to nerve ring and 119.5–134.5 μm from anterior end. Hemizonid visible, located 24.0–32.5 μm posterior to excretory pore or 144.5–164.0 μm from anterior end, rectum distinctly longer than anal body diameter; gubernaculum lanceolate shaped, ending in an elongated acute tip (Fig. 4C). The line drawings of Rühm (1956) show that corpus is longer than postcorpus. This character was used in the compendium of the genus presented by Andrássy (1983), to distinguish P. obtusa from related species. However, in the original description by Fuchs (1915), the corpus was noted to be equal in length to the postcorpus, which also agrees with the line drawings of Valizadeh et al. (2017). Additional details on the reproductive system include the female reproductive system being generally arranged from the anterior (distal) as ovary, oviduct, uterus junction, crustaformeria, uterus, vagina/vulva, extending 53–64% of total body length. Distal part of ovary reflexed dorsally, covering 33–55% of the total gonad system length. Oocytes arranged in two to three rows in the reflexed part of ovary. Oviduct composed of small ellipsoidal cells anteriorly and enlarged posteriorly with large round cells. Spermathecal-uterus junction visible. Uterus composed of irregularly arranged globular uterine cells, forming prominent crustaformeria, and the wider proximal tubular part with epithelial wall. Uterus often contains 1–3 developing eggs. Vagina short, oblique with muscular walls. Lips of vulva protuberate or not protuberate. Vulva a transverse slit. Male with a single testis, on right subventral region of intestine, extending 53–64% of total body length, anteriorly reflexed dorsally up to 17–27% of its length. Spermatocytes arranged in two to three rows in reflexed part. Vas deferens tube-like, occupying ca 25–28% of total gonad length, composed of large cells, joining the rectum and forming a narrow cloacal tube terminating into the cloaca.

The montaged two partially overlapping fragments of 18S rRNA gene sequences yielded ca 1700-bp long sequences for each studied species. No intraspecific sequence variation was recorded in the two newly obtained sequences (PQ589232, PQ589233) of Parasitorhabditis paraterebrana n. sp. In the 18S rRNA gene tree, sequences of Parasitorhabditis paraterebrana n. sp. were close to the few available sequences of other species of the genus. All sequences of Parasitorhabditis were grouped in a well-supported clade (PP=100). Notably, sequences of Parasitorhabditis paraterebrana n. sp. were highly similar to sequences of P. terebrana (OQ704209, OQ704209 and PQ589234), differing by only 4 bp (0.3%). Parasitorhabditis paraterebrana n. sp., sequences were also highly similar to the newly obtained sequences of P. obtusa (PQ589235, PQ589236), P. obtusa isolates accessioned EU003189, and KJ705089; and an unidentified Parasitorhabditis sp. (AF083028), differing by only 4–8 bp (0.4–0.5%). However, interspecific sequence differences of 31–33 bp (3.9–4.2%) were observed when compared with P. obtusa isolates accessioned MH590260 and the unidentified Parasitorhabditis spp. sequences (MH590259 and MH590261) from Taiwan.

The new 18S rRNA gene sequence for P. terebrana (PQ589234) was identical to P. terebrana sequences (OQ704209, OQ704209), with no intraspecific variation. The sequences obtained for P. obtusa (PQ589235, PQ589236) were 100% identical to those of P. obtusa isolate from Czech Republic (KJ705089); and highly similar to that of P. obtusa isolate accessioned EU003189 and the unidentified Parasitorhabditis sp. (AF083028), differing by only 3–4 bp (0.2%). The sequences also differed from short sequence data of P. obtusa isolate from Israel (MG865783) by 6 bp (0.6%); but significantly differed from the Taiwan isolate (MH590260) and the unidentified Parasitorhabditis spp. sequences (MH590259 and MH590261) by 31–33 bp (3.9–4.2%).

Amplification of the 28S rRNA gene yielded single amplicons of ca 600 bp. The three D2-D3 sequences from Parasitorhabditis paraterebrana n. sp. (PQ589220-PQ589222) showed no intraspecific variation. Similar to the 18S rRNA gene tree, the more informative 28S rRNA gene tree also showed close phylogenetic relationships among the available sequences of the genus, and all sequences of Parasitorhabditis are grouped in a well-supported clade (PP = 100%). Sequences of Parasitorhabditis paraterebrana n. sp. differed from P. terebrana sequences (OQ291289, OQ291290 and PQ589223) by 13 bp (2.4%); and from those of the closely similar species P. obtusa isolates (MF288651, MG865784, including the new sequences PQ589224, PQ589225), and two other more phylogenetically distant P. obtusa isolates (EF990724 and KM245037) by 19 bp (3.2–3.8%) and 25 bp (4.2–4.7%), respectively. The newly obtained sequences of P. terebrana (PQ589223) differed from the previously submitted sequences of P. terebrana (OQ291289, OQ291290) by 0–1 bp, and the obtained sequences of P. obtusa (PQ589224, PQ589225) differed from those of P. obtusa isolates (MF288651, MG865784, EF990724 and KM245037) by 7 bp (1.4%).

The amplified partial COI gene yielded single amplicons of ca 650 bp. There were only two COI sequences of Parasitorhabditis available in the GenBank database at the time of analysis (i.e., P. terebrana OQ281742 and OQ281743). The newly obtained sequence of P. terebrana (PQ585735) was 100% identical to the two aforementioned, available P. terebrana sequences. The new sequences of P. paraterebrana n. sp. (PQ585733, PQ585734) showed no intraspecific variation and differed from those of P. terebrana (OQ281742, OQ281743 and PQ585735) by 95–99 bp (15.4–15.4%). Parasitorhabditis paraterebrana n. sp. sequences (PQ585733, PQ585734) also differed from the newly obtained sequences of P. obtusa (PQ585736, PQ585737) by 86–93 bp (13.8–14.2%). The two sequences of P. obtusa (PQ585736, PQ585737) showed an intraspecific variation of 2 bp. In the reconstructed COI gene phylogeny, the three species were closely related and formed a well-supported subclade (PP=100) among other rhabditid species. The amplified partial ITS rRNA gene yielded single amplicons of ca 650–850 bp. Similar to the partial COI gene, there were only a few ITS rRNA gene sequences of Parasitorhabditis available in the GenBank database; all from one species (i.e., P. terebrana OQ305560-OQ305564). No intraspecific variation was recorded in the new sequences of P. paraterebrana n. sp. (PQ589226-PQ589228), differing from those of P. terebrana (OQ305560-OQ305564 and PQ589229) and P. obtusa (PQ589230, PQ589231) by 79–82 bp (12.6–12.9%) and 93–114 bp (14.1–16.2%), respectively. The newly obtained sequence data of P. terebrana (PQ589229) was 100% identical to the available P. terebrana sequences (OQ305560-OQ305564).

Fifty-eight 18S rRNA, forty-two 28S rRNA, and thirty-six COI gene sequences from various member species of Rhabditina Chitwood, 1933, inclusive of the newly obtained sequences and outgroups, constituted the sequence dataset for phylogenetic analyses. Phylogenetic relationships, as inferred from Bayesian analysis of the dataset with GTR + I + G substitution model, are shown in Figures 5–7.

Figure 5:

Bayesian tree inferred under the GTR + I + G model from 18S rRNA gene sequences of Rhabditina Chitwood, 1933 species. Posterior probability values exceeding 50 % are given on appropriate clades. The studied population is indicated in bold text. Outgroups: Rhabditoides inermiformis (Osche, 1952) Dougherty, 1955 and Rhomborhabditis regina (Schulte and Poinar, 1991) Sudhaus, 2011.

Figure 6:

Bayesian tree inferred under the GTR + I + G model from LSU D2-D3 partial sequences of Rhabditina Chitwood, 1933 species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroups: Rhabditoides inermiformis (Osche, 1952) Dougherty, 1955 and Rhomborhabditis regina (Schulte and Poinar, 1991) Sudhaus, 2011.

Figure 7:

Bayesian tree inferred under the GTR + I + G model from COI partial sequences of Rhabditina Chitwood, 1933 species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroups: Chylorhabditis epuraeae Kanzaki, Hamaguchi and Takeuchi-Kaneko, 2021, and Rhabditis Dujardin, 1844 species.