The genus Pseudacrobeles was erected to include a single species, Pseudacrobeles variabilis Steiner, 1938, which was formerly Acrobeles variabilis Steiner, 1936. During a taxonomic revision of the genus, De Ley et al. (1993a, 1993b) considered 12 species under two subgenera (Bunobus and Pseudacrobeles). The subgenera were distinguished from each other based on the following characters: for Pseudacrobeles, triradiate lip region (very rarely with hexaradiate or bilateral symmetry) and labial probolae present (if absent then lips low and amalgamated); for Bunobus, lip region bilaterally symmetric, labial and cephalic probolae absent and subdorsal and subventral lips larger than lateral lips. Recently, Andrássy (2005) elevated the subgenus Pseudacrobeles (Bunobus) to genus level separate from the subgenus Pseudacrobeles. However, until further confirmation, we here treat each as a subgenus.

Pseudacrobeles species have been reported from every continent in the world except Antarctica. The distribution of Bunobus and Pseudacrobeles greatly overlap. Both are found in South America (Loof, 1964; Andrássy, 1968; Zell, 1987; De Ley et al., 1993a, 1993b), Europe (Zell, 1987; Hernández, 1990; Holovachov and De Ley, 2001; Abolafia et al., 2002; Abolafia and Peña-Santiago, 2005; Holovachov and Boström, 2006; Abolafia and Peña-Santiago, 2013), southern and western Asia (De Ley et al., 1993a, 1993b; Shokoohi and Abolafia, 2012), and Africa (Schuurmans-Stekhoven, 1951; Loof, 1964; De Ley et al., 1993a, 1993b). Thus far only Pseudacrobeles has been described from North America (De Ley et al., 1993a) and Oceania (Boström and Holovachov, 2011). Despite their nearly worldwide distribution, Pseudacrobeles is unknown from northeast Asia, possibly due to lack of survey effort. A new species, described herein as Pseudacrobeles (Pseudacrobeles) curvatus sp. n., was isolated from soil samples collected from potato fields in South Korea. A detailed morphological description based on morphometric analyses including observations from light microscopy (LM) and scanning electron microscopy (SEM) is provided. As molecular barcode references, we also provide sequence information for the D2-D3 regions of 28S rDNA and the ITS1-5.8S-ITS2 region of rDNA.

Material and Methods

Nematode isolation and culture: Soil samples were collected from potato farms in Changwon-si, Gyeongsangnam Province, South Korea. Nematodes were extracted by the sieving and Baermann funnel method (Baermann, 1917). An in vitro culture was established from a single female specimen and maintained at room temperature (18–20°C) on soil agar plates containing 25 mg/ml autoclaved soil, 5 μg/ml cholesterol, and 1% agar.

Morphological observations: Individual nematodes were placed in 2 ml water in a 15 ml tube and quickly fixed with the addition of 4 ml of 80°C TAF (2% triethanolamine and 7% formaldehyde), then processed to dehydrated glycerin following Seinhorst (1959) and mounted in pure glycerin on HS-slides (Shirayama et al., 1993). Morphological characters were observed under an optical microscope (Olympus BX-51) with differential interference contrast (DIC). Morphometrics were performed on images obtained with a CoolSnap Photometrics color CCD digital camera and QCapture Pro 5.

Scanning electron microscope: Prior to SEM imaging, nematodes were fixed in TAF and held for at least 24 hr at room temperature, then post-fixed in a solution of 4% aqueous osmium tetroxide at 4°C for 3 d. Fixed specimens were dehydrated using a series of 10%–100% absolute ethanol for 1 hr at each concentration level and then dried in a Hitachi HCP-2 critical point drier. Dried nematodes were mounted on copper/nickel tape attached to SEM stubs. The stubs with mounted nematodes were sputter-coated with gold/palladium using a sputter-coater (Eiko IB-3) and the morphology of nematodes was observed under high-vacuum conditions using a Zeiss Ultra Plus at 15 kV.

Molecular procedures and sequence analyses: Total genomic DNA from the new species was extracted using an Epicentre MasterPure DNA Purification Kit (Epicentre Co.) according to the manufacturer’s protocol. The ITS1-5.8S-ITS2 region and the D2-D3 regions of 28S rDNA were PCR amplified using universal primer sets (TW81/AB28 [Joyce et al., 1994] for ITS1-5.8S-ITS2 region and D2A/D3B [De Ley et al., 1999] for the D2-D3 region in 28S). PCR reactions were performed in a total volume of 50 μl, containing 2 μl of template DNA, 10 pmol of each primer, 10× Ex Taq buffer, 0.2 mM dNTP mixture, and 1.25 U of Taq polymerase (TaKaRa Ex Taq). PCR amplification conditions included an initial denaturing step at 95°C for 1 min, 35 cycles with a denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN Co.) following the manufacturer’s instructions. PCR-amplified fragments were then sequenced using Big Dye Terminator Cycle-Sequencing (Applied Biosystems).

Sequences of the ITS1-5.8S-ITS2 region and the D2-D3 regions of 28S rDNA were aligned to their homologous gene sequences of some other Pseudacrobeles species available from GenBank, in Clustal X with default options (Thompson et al., 1997). Both ends of the aligned datasets were trimmed before sequence analyses were performed using the Genious 6 program.

Results

Pseudacrobeles (Pseudacrobeles) curvatus sp. n. (Figs. 1,2; Table 1)

Fig. 1.

Pseudacrobeles (Pseudacrobeles) curvatus sp. n. A. Whole female. B. Neck region. C. Female reproductive system. D: Female posterior region.

Fig. 2.

Pseudacrobeles (Pseudacrobeles) curvatus sp. n. (scanning electron microscopy). A. Lip region. B. Anterior region. C. Excretory pore (arrow). D. Vulva. E. Lateral field. F. Female tail (arrow indicates phasmid opening near the end of the lateral lines).

Description

Female: Body cylindrical, length 453.1 to 563.4 µm, usually ventrally curved after fixation but occasionally irregularly contorted. Cuticle annulated; annuli 1.6 to 2.3 µm wide and 0.7 to 1.3 µm at midbody level. Three lateral incisures, end posteriorly near phasmid opening level on the tail. Head region continuous with the neck. Lip region 5.4 to 6.9 µm wide, triradiate symmetry with 6+4 papillae. Cephalic probolae absent; three ridge-shaped, low rounded labial probolae present. Transverse, oval-shaped amphidial openings. Stoma cephaloboid, about 1.6 to 2.3 times length of the lip region diameter; bar, oval or comma-shaped cheilorhabdions. Cheilostom similar in width to the gymnostom and with a dorsal denticle on metastom. Pharyngeal corpus cylindrical, 2.8 to 3.1 times isthmus length. Isthmus narrower than corpus, and distinctly demarcated from corpus. Basal bulb oval-shaped with well-developed valve; cardia conoid, surrounded by intestinal tissue. Nerve ring at posterior end of corpus or boundary between corpus and isthmus, 47 to 59 annuli from the anterior end, at 58.3% to 65.2% of total neck length. Excretory pore at the level of posterior end of corpus to anterior end of isthmus, 50 to 62 annuli from anterior end, at 59.8% to 69.6% of total neck length. Deirid in the lateral field at level of isthmus, 55 to 67 annuli from anterior end, at 65.7% to 75.1% of pharynx length. Female reproductive system monodelpic-prodelpic. Vulva either protruding or not, postvulval sac 0.8 to 1.3 times the body width at the level of the vulva. Uterus tubular, 1.7 to 2.4 times the body diameter. Spermatheca 18.3 to 31.7 mm long. Oviduct very short. Ovary straight to posterior, sometimes with double flexure posterior to vulva, with a single row of oocytes. Rectum 1.2 to 1.8 times the anal body diameter. Tail ventrally curved and sigmoid elongated conoid, with 25 to 32 annules. Phasmid openings at 30% to 41.5% of tail length.

Male: Unknown.

Diagnosis and relationships: The new species P. curvatus sp. n. is characterized by having a small body (453.1–563.4 µm), three pairs of symmetrical lips, no cephalic probolae, pharyngeal corpus 2.8 to 3.1 times the isthmus length, nerve ring lying between corpus and isthmus, vulva position at 61.3% to 66.0% of total body length, uterus 39.3 to 59.0 mm long, spermatheca 18.3 to 31.7 µm long, postvulval uterine sac 0.8 to 1.3 times the body diameter in length, tail sigmoid elongated conoid (43.9–54.7 µm tail length, c = 9.6–10.6, c′ = 3.5–4.6), and phasmid at 30.2% to 41.5% of tail length.

The new species shows morphological characters typical of the subgenus Pseudacrobeles by having radial symmetry of the lips and three lateral incisures that fade posteriorly near the level of phasmid openings. However, this new species is distinguished from other Pseudacrobeles species by morphological characters such as the absence of cephalic probolae, the similar widths of the cheilostome and gymnostom, and several measurements (Table 2). The new species resembles P. (P.) variabilis (Steiner, 1936) Steiner, 1938; however, it is distinguished by a shorter and wider tail (c = 9.6–10.6 vs 5.4–9.6 and c′ = 3.5–4.6 vs 4.6–8.7) (De Ley et al., 1993a). Also, this new species differs from P. (P.) baloghi (Andrássy, 1968) De Ley, Siddiqi and Boström, 1993 by its shorter tail (c = 9.6–10.6 vs 7.1–9.2), the absence of cephalic probolae, and the relatively posterior position of the nerve ring (at the level of the corpus–isthmus junction vs three-fifths of the length of the corpus region) (De Ley et al., 1993a). Compared to P. (P.) pauciannulatus (Marinari-Palmisano, 1967) De Ley, Siddiqi and Boström, 1993, the new species is distinguished by 25 to 32 annuli on the tail (vs 20–24), the absence of cephalic probolae (vs present cephalic probolae), shorter cardia (2–4 vs 6.5 µm) and a gymnostom and cheilostome with similar widths (vs gymnostom narrower than cheilostome) (Rashid et al., 1984; Rashid et al., 1988). The new species resembles P. (P.) unguicolis Abolafia, Liébanas and Peña-Santiago, 2002, but it is distinguished by a shorter tail (43.9–54.7 vs 54–75 µm), tail with 25 to 32 annuli (vs 38–41), uterus 1.7 to 2.4 times the body diameter (vs 3 times) and shorter vagina (0.2–0.3 vs 0.5 times the body width). A detailed comparison of the morphometrics and diagnostic features of Pseudacrobeles species is shown in Table 2.

Table 2.

Morphometrics and diagnostic features of species of Pseudacrobeles (Pseudacrobeles) Steiner, 1938.

Type habitat and locality: Specimens of P. (P.) curvatus sp. n. were collected from a small potato field in Bukmyeon, Uichang-gu, Changwon-si, Gyeongsangnam Province, South Korea (GPS coordinates: N 35° 22′ 22.5′′, E 128° 36′ 47.3′′). The soil was taken from the potato farm during a period of rest after the harvest.

Type material: Holotype (slide no. KOSPIV0000243269) and two paratypes (slide no. KOSPIV0000234931 and KOSPIV0000234932) are deposited in the National Institute of Biological Resources, Republic of Korea. Six paratypes (slide no. 01010401004-01010401009) are deposited in the Animal Phylogenomics Laboratory, Ewha Womans University, South Korea.

Etymology: The specific epithet refers to having a bent tail.

Molecular characterization: Sequences of the D2-D3 region of 28S and the ITS1-5.8S-ITS2 region of rDNA were obtained from P. (P.) curvatus sp. n. (GenBank accession no. KX889090 and KX889083, respectively). Comparison with some other Pseudacrobeles species shows that the D2-D3 region sequence differs by 14.1% (Pseudacrobeles sp. JB-85, DQ145654 and Pseudacrobeles sp. OH-2016) and 16.2% (P. (b.) bostromi, HM439772) (Table 3) and the ITS1-5S-ITS2 region shows a 25.3% difference from Pseudacrobeles sp. JB056 (DQ146429). However, molecular sequence information of D2-D3 and ITS1-5S-ITS2 for other species belonging to the genus Pseudacrobeles is very limited: only six D2-D3 of 28S rDNA sequences (four of which are from unidentified species) and one ITS1-5S-ITS2 region sequence from an unidentified species are available on GenBank. Further molecular analyses from diverse Pseudacrobeles species will help to better understand the sequence diversity and genetic divergence level among the species of this genus.