The superfamily Cephaloboidea Filipjev, 1934, characterized with a monodelphic-prodelphic reproductive system with sac-like spermatheca, includes six families, three of which are divided in two subfamilies (Andrássy, 2005; Shokoohi and Abolafia, 2019) Alirhabditidae Suryawanshi, 1971 with a long tubular stoma and lacking labial probolae; Bicirronematidae Andrássy, 1978 having stoma with well-developed cheilostom and with labial cirri; Cephalobidae Filipjev, 1934 with short cheilostom including Acrolobinae De Ley, Siddiqi and Boström, 1993 without labial probolae and Cephalobinae Filipjev, 1934 with three labial probolae of variable morphology; Daubayliidae Chitwood and Chitwood, 1934 including a parasite of snails with a reduced stoma; Elaphonematidae Heyns, 1962a with irregular lips and reduced stoma including Elaphonematinae Heyns, 1962a with bilaterally symmetric lip region and Kirjanoviinae Andrássy, 1976 with three nearly triangular labial probolae; Osstellidae Heyns, 1962b with fused lips and poorly developed basal bulb including Osstellinae Heyns, 1962b with very short stoma and Drilocephalobinae (Ali, Suryawanshi and Chisty, 1973) with a nearly absent stoma.
In the present study, a new genus and species are described from Iran that has morphological and molecular features that are in between the families Cephalobidae and Elaphonematidae. Additionally, a related species,
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
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).
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
According to observations of SEM studies (Baldwin et al., 2001; Karegar et al., 1997, 1998; Susulovsky et al., 2001), the new genus
On the other hand, the morphology of the lip region resembles
The generic name refers to the presence of acute processes (Latin
Morphometrics of
Sex | Holotype | Paratypes | |
---|---|---|---|
|
Female | 11 females | 6 males |
L | 575 | 612.4 ± 34.8 (552–668) | 596.0 ± 25.3 (578–645) |
a | 21.3 | 21.0 ± 0.6 (20.0–22.0) | 24.1 ± 2.5 (20.2–27.9) |
b | 3.7 | 4.3 ± 0.1 (4.0–4.5) | 3.5 ± 0.2 (3.2-3.8) |
c | 14.0 | 15.4 ± 1.2 (13.8–17.7) | 14.5 ± 0.6 (13.9–15.7) |
c' | 2.3 | 2.0 ± 0.1 (1.8–2.3) | 2.0 ± 0.1 (2.0–2.2) |
V | 62 | 61.2 ± 0.9 (60–63) | – |
Lip region width | 9 | 5.5 ± 0.4 (5–6) | 5.1 ± 0.4 (5–6) |
Stoma length | 13 | 12.1 ± 0.4 (11–13) | 11.5 ± 0.5 (11–12) |
Corpus length | 73 | 70.6 ± 4.3 (63–76) | 88.1 ± 7.1 (81–101) |
Isthmus length | 46 | 45.2 ± 1.3 (44–48) | 45.3 ± 2.4 (42–49) |
Bulbus length | 25 | 25.9 ± 1.5 (24–28) | 24.3 ± 0.6 (24–25) |
Pharynx length | 144 | 141.8 ± 4.7 (134–148) | 157.8 ± 6.9 (150–168) |
Neck length | 157 | 154.0 ± 4.9 (146–161) | 169.3 ± 7 (162–180) |
Nerve ring-ant. end | 89 | 98.7 ± 8.9 (85–112) | 100.8 ± 9.1 (86–110) |
Excretorypore-ant. end | 94 | 104.2 ± 5.6 (94–113) | 100.2 ± 4.5 (95–106) |
Deirid-ant. end | 117 | 110.0 ± 4.2 (107–113) | 122.0 ± 3.6 (118–125) |
Cuticle thickness | 3 | 2.6 ± 0.1 (2–3) | 2.2 ± 0.2 (2–3) |
Annuli width | 4 | 5.1 ± 0.5 (4–6) | 5.0 ± 0.2 (4–5) |
Body width neck base | 26 | 27.6 ± 1.5 (26–30) | 23.8 ± 1.7 (21–27) |
Body width mid-body | 27 | 29.0 ± 1.7 (26–32) | 24.9 ± 2.7 (21–29) |
Lateral field width | 7 | 8.2 ± 0.8 (7–10) | 7.3 ± 0.5 (7–8) |
Ovary length | 120 | 122.0 ± 20.7 (96–142) | – |
Oviduct length | 9 | 11.2 ± 2.2 (8–13) | – |
Spermatheca length | 32 | 31.6 ± 7.1 (23–39) | – |
Uterus length | 70 | 68.2 ± 16.9 (43–79) | – |
Vagina length | 9 | 7.6 ± 0.7 (7–9) | – |
Post-vulval uterinesac | 29 | 25.7 ± 1.4 (23–28) | – |
Vulva-ant. end | 357 | 375 ± 25.8 (331–418) | – |
Rectum length | 23 | 19.4 ± 1.9 (17–22) | – |
Body width anus | 18 | 18.9 ± 1.1 (17-21) | 19.8 ± 0.6 (19–21) |
Tail length | 41 | 39.8 ± 2.6 (37–44) | 40.9 ± 0.6 (40–42) |
Phasmid-anus distance | 18 | 22.4 ± 10.4 (17–41) | 19.1 ± 2.4 (18–23) |
Spicules length | – | – | 24.5 ± 0.8 (24–26) |
Gubernaculum length | – | – | 12.4 ± 1.1 (11–14) |
Stout to moderately slender nematodes, body length 0.55–0.67 mm. Habitus slightly ventrally curved in females and J-shaped in males after fixation. Cuticle deeply tessellated, having deep transversal and longitudinal incisures dividing the cuticle in blocks, the first annuli with wider than long blocks, later quadrangular and posteriorly longer than wide until the tail end. Lateral field with three longitudinal incisures or two narrow and tessellated wings, occupying 26–31% of mid-body diam., extending to tail end. Anterior body end narrower with lip region continuous with adjacent body, having six lips, slightly triangular, the lateral ones larger, and bearing six smaller labial and four larger cephalic sensilla; primary axils deep, V-shaped, bearing two conoid-elongate guard processes originating from each lip; secondary axils deep, U-shaped, with one thorn-like process (labial probolae?) in lateral view bearing a small rounded protuberance fused at an oral plate having triradiate symmetry developing more acute margin toward each primary axil. Amphid openings clearly visible, large, rounded to slightly oval. Oral opening hexagonal. Stoma cephaloboid with cheilostom bearing minute and rounded rhabdia, gymnostom very short, stegostom with minute discernible rhabdia being prostegostom longer and metastegostom bearing a minute dorsal tooth. Pharynx cephaloboid with pharyngeal corpus subcylindrical with metacorpus not well differentiated, isthmus unusually slender, 1.4–1.7 times corpus length, basal bulb pyriform, with well-developed valvular apparatus (grinder). Cardia conoid, surrounded by intestinal tissue. Nerve ring at 53–70% of neck length, surrounding the isthmus. Excretory pore at 58–74% of neck length, situated at level of isthmus, 18–25 annuli from anterior end. Deirids poorly discernible, at 72–75% of neck length, situated at level of isthmus, 23–29 annuli from anterior end. Intestine without distinct specialization.
Reproductive system cephaloboid, monodelphic-prodelphic having a globular sac-like spermatheca, in dextral position to intestine; ovary short, lacking flexures, with oocytes in one row; oviduct very short, areolate; spermatheca well-developed, as width as corresponding body diam., sometimes with sperm, this with 3–4 µm long; uterus about three times as long as corresponding body diam., distally tubular with thick walls and proximally swollen with thin walls; post-vulval uterine sac poorly developed, 0.8–1.0 times body diam., proximally swollen with thin walls and distally narrower lacking lumen; vagina short, extending inward 22–28% of body diam.; vulva very reduced, oval. Rectum 0.8–1.3 times anal body diam., with three small gland-like cells distinguishable around intestine-rectum junction. Tail conoid with small rounded terminus, with 9–12 annuli at ventral side. Phasmids located at 43–47% of tail length.
General morphology similar to female. Reproductive system monorchic, dextral in position, with testis reflexed ventral anteriorly. Spicules paired and symmetrical, with rounded, ventral bent manubrium, conoid calamus and slightly ventrally curved lamina with very small dorsal hump, poorly developed ventral wing and acute tip. Gubernaculum well developed, ventrally curved, 0.5–6.0 times the spicule length, with thin manubrium and corpus, and crura well developed. Three small gland-like cells distinguishable around beginning of cloaca. Genital papillae one pair pre-cloacal, one pair ad-cloacal and five pairs post-cloacal arranged as follows: two in middle tail region (one lateral located at lateral field level and one subventral), and three pairs near tail terminus (one subdorsal, one lateral, and one subventral). Tail conical and ventrally curved with small rounded terminus. Phasmids located at 43–55% of tail length, slightly posterior to genital papillae GP3.
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
The specimens were collected at sandy soil in the rhizosphere of
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
The morphological analysis of
Molecular analysis based on 18S rDNA (Fig. 5) does not resolve well the phylogenetic relationships of
Other species with an irregular lip pattern is
According to this,