Description of Spinocephalus tessellatus n. gen., n. sp. (Rhabditida, Cephalobidae) from Iran, a nematode with a new morphological pattern at lip region

The nematodes were extracted with a modified Baermann tray method (Whitehead and Hemming, 1965), killed and fixed by hot FPG (4:1:1, formaldehyde: propionic acid: glycerol), processed to anhydrous glycerol (Grisse, 1969), and mounted on glass microscope slides.

Photomicrographs were taken with a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with a differential interference contrast (DIC) optics mounted with a Nikon Digital Sight DS-U1 camera and processed with Adobe^® Photoshop^® CS. Demanian indices (de Man, 1881) and other ratios were calculated. The terminology used to describe the morphology of stoma and spicules-gubernaculum follows De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

Specimens preserved in glycerine were selected and prepared for observation with a SEM according to Abolafia (2015). They were cleaned 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).

Nematode DNA was extracted from single individuals, previously fixed in 70% ethanol, using a modified DNA extraction and PCR assays described by Castillo et al. (2003) somewhat modified (Archidona-Yuste et al., 2016). The specimens were cut in small pieces using the acute tip of a sterilized dental anesthesia needle on a clean slide with 18 ml of TE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), transferred to a microtube and adding 2 μl proteinase K (700 μg/ml^‒1) (Roche, Basel, Switzerland), and stored to –80°C within 15 min (for several days) until processing. Finally, the microtubes were incubated at 65°C (1 hr), then at 95°C (15 min) and the solution were use as DNA template. For DNA amplification, 3 μl of the extracted DNA was transferred to a microtube containing: 0.6 μl of each primer (10 mM), 3 μl Master Mix Taq DNA Polymerase (5x Hot FirePol Blend Master Mix) and ddH2O to a final volume of 20 μl. The primers used for amplification of the region of 18S rRNA gene were the forward primer SSU F_04 (5′-GCTTGTCTCCAAAGATTAAGCC-3′) and the reverse primer SSU R_26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Blaxter et al., 1998). The primers used for amplification of the D2-D3 region of 28S rRNA gene were the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (De Ley et al., 1999; Nunn, 1992). PCR cycle conditions were as follows: one denaturation cycle of 94°C for 15 min., followed by 35 cycles of 94°C for 45 sec; annealing cycle of 55°C for 45 sec; extension cycle of 72°C for 45 sec, and finally one extension cycle of 72°C for 5 min. After DNA amplification, 5 μl of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V–2, Progen Scientific, London, UK). The bands were stained with RedSafe (20,000x) previously added to the agarose gel solution. The sequencing reactions of the PCR products were performed at Sistemas Genómicos (Paterna, Valencia, Spain) according the Sanger et al. (1977) method. The rDNA sequences obtained for Spinocephalus tessellatus n. gen., n. sp. were submitted to the GenBank database.

For phylogenetic relationships, analysis was based on 18S and 28S rDNA. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with other 28S rRNA gene sequences available in GenBank using ClustalW (Thompson et al., 1994) alignment tool implemented in the MEGA7 (Kumar et al., 2016). Alignments ends were trimmed using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012). A phylogenetic trees were generated with the Bayesian inference method using MrBayes 3.2.6 (Ronquist et al., 2012). Drilocephalobus sp. (AY284680) for the 18S tree and Deficephalobus desenderi De Ley and Coomans, 1990 (GU062820) for the 28S tree was chosen as the outgroup. The analysis under GTR + I + G model was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) (Larget and Simon, 1999) for 1 × 10⁶ generations. The tree was visualized and saved with FigTree 1.4.4 (Rambaut, 2018).

Spinocephalus n. gen.

Cephalobidae, Cephalobinae. Cuticle with tessellations, lateral field with two tessellated wings. Lip region with six triangular lips bearing two conoid-elongate processes at primary axils and one thorn-like process (labial probolae?) at secondary axils bearing a small rounded protuberance fused at oral plate with triradiate symmetry, oral opening surrounded by a hexagonal margin, amphids large and clearly rounded to slightly oval, stoma cephaloboid with cheilostom bearing minute and rounded rhabdia. Pharynx cephaloboid with corpus subcylindrical and isthmus unusually very long. Nerve ring and excretory pore at isthmus level. Female monodelphic-prodelphic with spermatheca well developed and post-vulval uterine sac poorly developed. Female tail conoid with rounded terminus. Male monorchic with spicules paired and symmetrical and gubernaculum well-developed. Male tail conical and ventrally curved with rounded terminus.

The new genus Spinocephalus n. gen. resembles, morphologically, other genera of the superfamily Cephaloboidea that have cuticle divided in blocks and arranged into longitudinal crests such as Acromoldavicus Nesterov, 1970, Penjatinema Heyns and Swart, 1998, and Stegelleta Thorne, 1938.

According to observations of SEM studies (Baldwin et al., 2001; Karegar et al., 1997, 1998; Susulovsky et al., 2001), the new genus Spinocephalus is distinguished from Acromoldavicus by having lips divided into plates with one long, acute process at primary axils and one large thorn-like process curved toward the oral opening at secondary axils (vs expanded lips with acute tips at primary axils and rounded at secondary axils), oral opening surrounded by a triacute margin with tips directed toward the primary axils and bearing three small rounded protuberance, one dorsal and two subdorsal (vs surrounded by three triangular labial probolae). Likewise, the new genus is different from Penjatinema (Heyns and Swart, 1998; Holovachov et al., 2009) by the lip region morphology (vs lips with fimbriated margin and oral opening surrounded by three long labial probolae with dendriform distal part). Finally, Spinocephalus n. gen. can be differentiate from Stegelleta (SEM by Boström and Holovachov, 2012, 2014; Orselli and Vinciguerra, 2002) also by the lip region morphology (vs fused lips in pairs with smooth margin or having short acute tip at primary axils and oral opening surrounded by three bifurcate labial probolae with smooth prongs. Molecularly, only Acromoldavicus presents closer relationships with Spinocephalus n. gen.

On the other hand, the morphology of the lip region resembles Chilodellus Boström and Holovachov, 2012 with irregular lips and unusually large amphids. However, labial probolae are very different, having bifurcate distal halves with convergent prongs in Chilodellus. Unfortunately, molecular studies are not available to confirm this relationship.

The generic name refers to the presence of acute processes (Latin spina = thorn) on the lip region (Latin cephalus from Greek kephale = head).

Spinocephalus tessellatus n. gen., n. sp.

(Figs. 1–4 and Table 1).

Figure 1:

Figure 2:

Figure 3:

Figure 4:

A sequence with 6,258 bp (MZ621174) of the 18S rDNA and two sequences lacking differences with 936 bp (MZ621172, MZ621173) of the 28S rDNA fragment were obtained for Spinocephalus tessellatus n. gen., n. sp. This genus and species show a higher similarity with some species of the genera Acromoldavicus and Nothacrobeles Allen and Noffsinger, 1971 maintain a common aligned fragment with 657 bp. With respect to Nothacrobeles abolafiai Mehdizadeh and Shokoohi, 2019 (KC182515) the common 28S fragments show 52 bp (7.9%) differences (substitutions, deletions, or insertions), 72 bp (11.1%) differences with N. cancellatus (Thorne, 1925) Ruiz-Cuenca and Abolafia, 2020 (HM439765) and 137 bp (20.8%) differences with N. hebetocaudatus Abolafia, Divsalar, Panahi and Shokoohi, 2014 (KJ508411). With respect to Acromoldavicus mojavicus (AY027536, DQ145626) shows 66 bp (10.0%) differences, while with A. skrjabini (AY027535) shows 75 bp (11.4%) differences.

The specimens were collected at sandy soil in the rhizosphere of Tamarix passerinoides Delile ex Desv. in Shush (ancient Persian city of Susa, GPS coordinates: 32°17.28′N, 48°25.07′E), Khuzestan province, Iran.

Six females (holotype and paratypes) and five males (paratypes) deposited in the nematode collection of the Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Spain. One female and one male (paratypes) deposited in the nematode collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran.

The body length of the new species range from 0.55 –0.67 mm long in females and 0.58–0.65 mm long in males. The cuticle shows deep transversal and longitudinal incisures which divides the cuticle in blocks, or tessellated, and the lateral fields with two tessellated longitudinal wings or three longitudinal incisures. The lip region has six triangular lips appearing the primary axils deeper with V-shaped bearing two conoid-elongate guard processes originating from each lip while the secondary axils are deeper with U-shaped bearing one thorn-like process (labial probolae?) observed in lateral view and bearing a small rounded protuberance fused at an oral plate having triradiate symmetry which develops a more acute margin toward each primary axil. The oral opening is hexagonal The amphids are large and rounded to slightly oval. The stoma is cephaloboid with cheilostom bearing minute and rounded rhabdia. The pharynx is cephaloboid with subcylindrical corpus and very long isthmus being 1.4–1.7 times corpus length. The nerve ring surrounds the isthmus and the excretory pore appears at 65–73% of neck length at isthmus level. The female reproductive system is monodelphic-prodelphic with spermatheca as long as the body diam. and post-vulval uterine sac 0.8–1.0 times body diameter. The female tail is conoid with small rounded terminus. The male reproductive system is monorchic having spicules 24–26 µm long and gubernaculum 11–14 µm long. The male tail is conical and ventrally curved ending in a small, rounded terminus.

The specific name refers to the presence of cuticular blocks or tessellation (Latin tessella = mosaic pavers). Zoobank code urn:lsid:zoobank.org:pub:53D4EE5A-A6B4-4A91-9896-88A5A6134D6C

The morphological analysis of Spinocephalus tessellatus n. gen., n. sp. shows the presence of an unusual lip pattern which structure is not similar to other genera belonging to the superfamily Cephaloboidea. The presence of a stoma with small rhabdia and sac-like spermatheca clearly indicates its relationship with other species of the infraorder Cephalobomorpha. However, the absence of clear labial probolae and the unusual structure of the lips make difficult to know its position at family level.

Molecular analysis based on 18S rDNA (Fig. 5) does not resolve well the phylogenetic relationships of Spinocephalus n. gen. because few genera of the superfamily Cephaloboidea are sequenced currently, appearing the new genus grouped to some species of the genus Acrobeles von Linstow, 1877 although they do not maintain closemorphological similarities. On the other hand, the 28S rDNA phylogenetic tree (Fig. 6) shows that the new genus is related with species of the genera Nothacrobeles and Acromoldavicus, both containing species with tessellated cuticle such as N. cancellatus (see Karegar et al., 1997, 1998) and Acromoldavicus species (Fig. 7). With respect to the lip region, the lip pattern of Spinocephalus n. gen. (Fig. 8A) resembles slightly to that in Acromoldavicus (Fig. 8B). Thus, the ventral triangular process of Acromoldavicus could be homologous to the polygonal plate appearing in Spinocephalus n. gen., while the elongate process visible at the primary axils could be homologous to the acute tip of each lip present in Acromoldavicus. However, the lip pattern observed in the Nothacrobeles species, with dentate lips, is very different.

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Other species with an irregular lip pattern is Chilodellus eremus Boström and Holovachov, 2012 (Fig. 8C). This species has very large amphid openings, similar to Spinocephalus tessellatus n. gen., n. sp., and lips with long, acute processes, the lateral ones more reduced, which have a large amphid opening. However, the labial probolae have very different morphology with bifurcate distal part with pinnate outer margin.

According to this, Spinocephalus tessellatus n. gen., n. sp. is tentatively located in the family Cephalobidae, subfamily Cephalobinae instead of the family Elaphonematidae, subfamily Kirjanoviinae.