Introduction

Nematodes of the order Rhabditida are an important group in the phylum Nematoda and can occupy different ecological niches (Shokoohi and Abolafia 2019). This group has a significant environmental role (mineralization of soil, pathogenicity on insects, the transmission of bacterial pathogens of vegetables, feed on bacteria, etc.). The taxonomy of these nematodes was revised by different authors (Andrássy 2005; Blaxter et al. 1998; De Ley and Blaxter 2002; Sudhaus and Fitch 2001; Shokoohi et al. 2007; 2008; Shokoohi and Abolafia 2019). This order contains numerous species (Andrássy 2005).

One of these species is Poikilolaimus oxycercus (de Man 1895) Sudhaus 1980, described previously from South Africa as Cuticularia mathesoni by van der Linde (1938) and, more recently, reported by Borgonie et al. (2015).

Furthermore, Pseudacrobeles (Pseudacrobeles) macrocystis De Ley and Siddiqi 1991 was described by De Ley and Siddiqi (1991) from Malaysia, providing SEM study. Later, this species was redescribed by De Ley et al. (1993a) from Brazil, Cameroon, and Tanzania, providing a new SEM study of the lip region and male posterior end. Then, Shokoohi and Abolafia (2012) described this species from Iran. Although this species was not found previously in South Africa, other species of this genus, Pseudacrobeles elongatus (de Man, 1884) Abolafia and Peña-Santiago 2005 (as Cephalobus elongatus de Man, 1884), was described in South Africa by van der Linde (1938).

Several years ago, we started a research project on the identification and biodiversity of free-living and plant-parasitic nematodes in South Africa. The present paper deals with two known species belonging to the families Poikilolaimidae Dougherty 1955 and Cephalobidae Filipjev 1934 (taxonomic classification according to Shokoohi and Abolafia, 2019) collected in natural areas from South Africa. Therefore, the study aimed to (i) study Poikilolaimus and Pseudacrobeles using morphological and morphometrical analyses and (ii) study Pseudacrobeles using molecular analysis.

Materials and methods

Nematode extraction and processing

Nematodes were collected from wild grass soil samples and extracted from them using the Baermann (1917) funnel technique. Extracted individuals were fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin utilising the method of De Grisse (1969). Fixed specimens were mounted on permanent glass slides (see methodology provided by Abolafia (2022).

Light microscopy (LM)

Observations were made using a Leitz Laborlux S (Leitz, Wetzlar, Germany), a Nikon Eclipse 80i (Nikon, Tokyo, Japan), and a Zeiss (Axio Lab, Germany) microscope. Measurements (see methodology provided by Abolafia (2023) were taken with the Leitz and Zeiss (Axio Lab) microscopes, which have a drawing tube (camera lucida) attached to it, and the Demanian indices (de Man 1881) and other ratios were calculated. Drawings were made using a camera attached to a Zeiss microscope (Axio Lab, Germany), whereas LM pictures were taken with the Leitz microscope and the Nikon microscope, which is equipped with differential interference contrast (DIC) optics and a Nikon Digital Sight DS-U1 camera. Micrographs were combined using Adobe® Photoshop® CS. The terminology used for the morphology of stoma, spicules, and gubernaculum follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017).

Scanning electron microscopy (SEM)

Specimens preserved in glycerine were selected for observation under SEM, according to Abolafia (2015). They were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

DNA extraction, PCR, and sequencing

DNA was extracted from fresh nematodes following Holovachov et al. (2009). The specimens were picked using a fine-tipped needle and transferred together to a 1.5 ml capacity microtube containing 25 μl of double distilled water. The presence of the specimens in the tube was verified using a Zeiss microscope at the Aquaculture Research Unit. The nematodes were crushed with a sterile needle, after which 20 μl of 5% Chelex-100 (Sigma, USA) solution and 5 μl proteinase K (20 mg ml–1) were added to the nematode substrate. The homogenate was incubated at 56 °C for 2 h and then at 95 °C for 10 min (Shokoohi 2022). The supernatant was extracted from the tube and stored at – 20 °C. Partial 18S rDNA sequences were amplified using polymerase chain reactions (PCR) with the forward primer SSU_F_04 (5'-GCTTGTCTCAAAGATTAAGCC-3') and reverse primer SSU_R_26 (5'-CATTCTTGGCAAATGCTTTCG-3') (Blaxter et al. 1998). In addition, the 28S D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5′-TCGGAAGGAACCAGCTACTA-3') primers were used for the D2-D3 segment amplification (De Ley et al. 1999). The PCR reaction was done using 8 μl of nematode DNA extract, 8 μl of nuclease-free ddH2O, 12.5 μl ready to use Master Mix including Taq polymerase (Promega, USA), 1 μl of each of the two primers (10 pmol μl−1) to a final volume of 30.5 μl. The amplification was done using an Eppendorf Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany) with the following conditions: initial denaturation at 94 °C for 3 min, followed by 37 cycles entailing denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min. The final extension was achieved at 72 °C for 6 min. (Koohkan et al. 2015 and Shokoohi et al. 2023) for 18S rDNA and 28S rDNA, respectively). Following DNA amplification, 5 μl of PCR product was loaded in a 1.5% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with SafeView (abm, Canada) and visualised and photographed on a UV transilluminator. The DNA product was stored at – 20 °C until sequencing commenced. PCR products were sent to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa) for purification and sequencing. The obtained sequences were deposited in NCBI GenBank under accession numbers MW298529 (18S rDNA) and MW301158 (28S rDNA) for P. (P.) macrocystis.

Phylogenetic analyses

The newly obtained 18S and 28S sequences were manually edited using Chromas 2.6.6 (Technelysium, Queensland, Australia) and aligned with other 18S or 28S rDNA sequences available in GenBank using ClustalW as implemented in MEGA7 (Kumar et al. 2016). Poorly aligned regions at either end were trimmed using MEGA7. The model of nucleotide substitution was statistically selected using jModelTest 2.1.10 (Darriba et al. 2012). Phylogenetic trees were generated with the Bayesian inference method using MrBayes 3.2.6 (Ronquist et al. 2012). Aphelenchus avenae (JQ348399) and Teratolobus sp. (KJ652552) were chosen as outgroups for 18S and 28S rDNA phylogenetic analysis. The General Time Reversible Plus Invariant sites plus Gamma distribution (GTR + I + G) model was selected with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) analysis (Larget and Simon 1999) for 1 × 106 generations. The resulting trees were visualised and saved with FigTree 1.4.4 (Rambaut 2018).

Results

Poikilolaimus oxycercus (de Man 1895) Sudhaus 1980 (Fig. 1).

Fig. 1
figure 1

Poikilolaimus oxycercus (de Man 1895) Andrássy, 1983. A Neck. B Lip region. C Entire male. D Entire female. E Female reproductive system. F Female posterior end. G Male posterior end

Full size image

Material examined

Five females and five males in good condition.

Measurements

See Table 1.

Table 1 Measurements of Poikilolaimus oxycercus (de Man 1895) Andrássy, 1983 from South Africa
Full size table

Female

Body almost straight with slightly curved ventrad after fixation. Cuticle almost smooth, sock-like, 4.6–5.2 µm, finely annulated; annuli 1.2–1.3 µm wide. Lateral fields with one longitudinal line. Lip region is continuous with the neck, bearing small papillae. Amphids aperture is not visible. Stoma rhabditoid, with distinct cheilo-, gymno- and stegostom; cheilostom poorly cuticularised, gymnostom longer than cheilostom, with well-cuticularized walls; stegostom tubular at promesostegostom and with isomorphic and isotopic glottoid apparatus at metastegostom, bearing 1–2 denticles; telostegostom very short; gymnostom and promesostegostom fused, tubular; pharyngeal collar present surrounding the metatelosgostom. Pharynx rhabditoid; pharyngeal corpus, about 1.9–2.6 times isthmus length, with subcylindrical procorpus and slightly swollen metacorpus; isthmus robust, well distinguished from metacorpus; basal bulb spheroid, with prominent grinder with visible posterior haustrulum, this 16–20 µm long. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 66–68% of neck length. Secretory-excretory pore at isthmus level, at 79–80% of neck length. Deirids not visible. Intestine without distinct specialisation at anterior cardiac part. Reproductive system didelphic-amphidelphic; ovaries dorsally reflexed, lacking posterior flexures; oviducts short, tubular, not well differentiated from the ovary, differentiated posteriorly in a spheroid to oval-shaped spermatheca, with round sperm; uteri tubular, tubular with narrow lumen at the anterior part and swollen with wider lumen at the posterior part; vagina with thin walls, about one-third of the body diameter long; vulva with simple lips, located slightly postequatorial. Rectum 0.7–1.4 times anal body diameter. Tail slightly cupola shaped, with rounded anterior part and conoid posterior part. Phasmids located at base of the rounded part of tail.

Male

General morphology is similar to that of females. Body straight, slightly curved. Genital system monorchic, with testis ventrally reflexed. Tail cupola shaped with acute terminus. Bursa peloderan with eight genital papillae. Genital papillae comprise three pre-cloacal pairs and five post-cloacal pairs, in arrangement 1 + 2/1 + 1 + 3. Spicules free, ventrally curved, 1.3–1.5 times than the corresponding body diameter; manubrium rounded, hook-shaped, offset from calamus, calamus short, lamina ventrally curved with developed velum. Gubernaculum 29–38% of spicule length.

Remarks

Morphology and measurements of the South African population agree with those of the previous material examined (de Man 1895; van der Linde 1938; Sudhaus 1980; Andrássy 1983; Tahseen et al. 2009; Shokoohi et al. 2014; Kang et al. 2019); however, it differs in female tail length (24–35 vs 30–60 µm), and gubernaculum length (10–12 vs 10–16 µm). From Tahseen et al. (2009), population differs by having a slightly shorter female (vs 602–815 µm). Compared with the population studied by Shokoohi et al. (2014), they differ in body length (vs 574–677 µm in females and 487–675 µm in males), tail length (24–35 vs 13–20 µm in females and 40–43 vs 24–28 µm in males). Compared with the South Korean population (Kang et al. 2019), they differ in body length (vs 0.5–1.1 mm in females and 0.5–1.2 in males).

Pseudacrobeles (Pseudacrobeles) macrocystis De Ley and Siddiqi 1991 (Figs. 2, 3).

Fig. 2
figure 2

Pseudacrobeles (P.) macrocystis De Ley and Siddiqi 1991 (light microscopy). A Neck (black arrow pointing to the excretory pore, a white arrow pointing to the deirid); B anterior end; C female posterior end (arrow pointing to the phasmid); D entire female; E female reproductive system; F entire male; G lateral field; H male posterior end (black arrows pointing the genital papillae, white arrow pointing the phasmid)

Full size image
Fig. 3
figure 3

Pseudacrobeles (P.) macrocystis De Ley and Siddiqi 1991 (scanning electron microscopy). A, B Lip region in lateral and frontal views, respectively (red line surrounding one of the labial processes); C excretory pore (arrow); D, E Vulva; F Lateral field; G, J: anus in ventral and lateral views, respectively; H, I female posterior end in lateral and ventral views, respectively (arrow pointing the phasmid); K female phasmid (arrow)

Full size image

Material examined

Ten females and three males are in a good state of preservation.

Measurements

See Table 2.

Table 2 Measurements of Pseudacrobeles (Pseudacrobeles) macrocystis De Ley and Siddiqi (1991) from South Africa
Full size table

Description

Adult

Body 0.61–0.78 mm long. Habitus ventrad curved after fixation, C-shaped in female and J-shaped in male. Cuticle 2–3 µm thickness, with transverse striations; annuli 1–3 μm wide at midbody. Lateral field 4–5 µm wide, occupying 13–16% of midbody diameter, with two alae limited by three longitudinal incisures extending to the phasmids. Lip region with six conoid lips bearing an elongate conoid process; primary axils narrow and smooth, U-shaped, lacking guarding processes; secondary axils width and smooth, V-shaped lacking guarding processes. Labial probolae three, blunt conoid. Stoma cephaloboid; cheilostom with small comma-shaped rhabdia; gymnostom very short with rhabdia poorly discernible; stegostom muscular, elongate, with well-sclerotized rhabdia. Pharynx cephaloboid; pharyngeal corpus subcylindrical, elongate, 3.0–4.0 times isthmus length; isthmus more slender; basal bulb ovoid, bearing well-developed valves. Cardia conoid. Nerve ring at 61–80% of neck length, surrounding pharyngeal corpus at its posterior part. Excretory pore at 68–71% of neck length, at the level of the posterior part of the pharyngeal corpus. Deirids at 75–80% of neck length, at the level of the anterior part of the isthmus.

Female

Reproductive system monodelphic-prodelphic; ovary posteriorly directed, with double flexure posterior to vulva; oviduct short, alveolated; spermatheca well developed, 0.4–1.1 times body diam., with spermatozoa inside; uterus cylindrical, 2.1–3.2 times body diameter long, differentiated in a long distal part with a scarce lumen and thick walls, and a short proximal portion with thinner walls and distinct lumen; post vulval uterine sac 0.6–1.1 times the corresponding body diam. long; vagina straight, 27–41% of body-wide; vulva slightly protruding. Rectum 1.1–2.2 times anal body width long, with three rectal glands. Tail conoid-elongate with acute terminus. Phasmids at 23–35% of tail length.

Male

Reproductive system monorchic. Testis reflexed ventrally anteriorly. Tail ventrally curved, conoid, bearing a thin mucro with acute terminus. Genital papillae three pairs pre-cloacal and five pairs post-cloacal, one anterior subventral, one anterior lateral, two posterior ventral and one posterior subdorsal. Phasmids located posterior to the lateral genital papillae, at 27–33% of tail length. Spicules are ventrally curved, with rounded manubrium, wide calamus, and curved lamina with finely rounded tip. Gubernaculum ventrally curved, 0.5–0.6 times the spicules length, with thin manubrium and corpus with low central cuneus and well-developed lateral crura.

Remarks

The population examined of P. (P.) macrocystis from South Africa fits well with the original description of this species published by De Ley and Siddiqi (1991) from Malaysia, except for the female body length slightly longer (0.6–0.7 mm vs 0.5–0.6 mm), neck length (168–202 µm vs 153–174 µm), and tail (70–85 µm vs 57–76 µm). Besides, there are no significant differences between the populations studied by De Ley et al. (1993a) from Cameroon and Tanzania. Compared with the Iranian specimens of P. (P.) macrocystis examined by Shokoohi and Abolafia (2012), there are no critical differences, although the male was not reported for the Iranian population. This species is reported for the first time from South Africa.

Molecular characterisation

The nblast result of the 18S rDNA gene fragment of the South African population of P. (P.) macrocystis shows a relationship with other species of the genus with 13 different gaps (99% similarity) with Pseudacrobeles sp. (KU180672), 20 differences (98% similarity) with Pseudacrobeles (Pseudacrobeles) variabilis (AF202150).

Locality and habitat

Both species were recovered from Magoebaskloof (GPS coordinates: S: 23°52′40.368"; E: 29°56′14.459"), Limpopo Province, South Africa, in association with wild grass.

Voucher material

Eight females and three males of P. (P.) macrocystis were deposited in the nematode collection of the Aquaculture Research Unit of the University of Limpopo, South Africa, and two females were deposited in the nematode collection of the University of Jaén, Spain. In addition, two slides of P. oxycercus containing five females and five males were deposited at the Nematology collection of the Aquaculture Research Unit, University of Limpopo, South Africa.

Phylogenetic relationships of Pseudacrobeles (Pseudacrobeles) macrocystis

The phylogenetic analysis of the 18S (Fig. 4) and 28S (Fig. 5) rDNA trees agree with the traditional arrangement of the genera of the subfamily Cephalobinae based on morphological characters (Andrássy 2005), agreeing with other molecular studies (Abolafia et al. 2020, 2021). Thus, the genus Pseudacrobeles Steiner 1938 appears more basal, close to the genus Eucephalobus Steiner 1936, having poorly developed lips and labial probolae (plesiomorphic condition), while genera with complex lips and labial probolae (apomorphic condition) appear in the derived branches in both trees. According to the phylogenetic trees, the evolutionary relationships of Pseudacrobeles appear more or less clear.

Fig. 4
figure 4

Bayesian Inference tree from the newly sequenced Pseudacrobeles (P.) macrocystis De Ley and Siddiqi 1991 based on sequences of the 18S rDNA region. Bayesian posterior probabilities are given for each clade

Full size image
Fig. 5
figure 5

Bayesian Inference tree from the newly sequenced Pseudacrobeles (P.) macrocystis De Ley and Siddiqi 1991 based on sequences of the 28S rDNA region. Bayesian posterior probabilities are given for each clade

Full size image

Regarding the genus Pseudacrobeles, it was established by Steiner in 1938. Then, the genus Pseudacrobeles divided into two subgenera, including Pseudacrobeles Steiner 1938 and Bunobus De Ley et al. 1993a, b (De Ley et al. 1993a, 1993b). The subgenera differ in the morphology of the lip region. In subgenus Pseudacrobeles, cephalic probolae (setae-like) and labial probolae (conical or low ridges connecting the tips of adjacent lips) are present. Besides, lateral lips are well developed. However, in subgenus Bunobus, cephalic probolae, and labial probolae do not exist. In addition, lateral lips are reduced (see Shokoohi and Abolafia 2012). In conclusion, Pseudacrobeles (P.) macrocystis was observed in various soil types associated with multiple crops in South Africa. This species is bacterivorous; therefore, its ecological role in the soil and its effect on crop production needs to be investigated.