Morphological and molecular characterisation of Tripylina gorganensis from the Slovak Republic as a contribution to the redescription of the species

In total, ten representative soil samples composed from five subsamples were collected from the five permanent research plots (10 × 10 m) established in the undisturbed natural beech forest in Opátka (48°47’N, 21°04’E, at 776 m a.s.l.), a small village in Košice district, eastern Slovakia. Samples were collected twice, five in May 2016 and five in September 2018, and examined in two laboratories, first in Slovakia and later in Poland.

Each sample (collected in 2016) was homogenized by gentle hand mixing. A total of 100 g of soil was then soaked in 1 l of tap water for 30–60 min and processed by the Cobb sieving and decanting method (Cobb, 1918) followed by the modified Baermann technique (van Benzooijen, 2006). Subsequently, nematodes were extracted from the aqueous soil suspensions using a set of two cotton-propylene filters. Suspensions containing nematodes were collected after 24 h. The nematode suspensions were subsequently examined under a stereomicroscope (40 × and 60 × magnification). After the excess water was removed, five Tripylina individuals was set aside for molecular analyses and therefore placed in an Eppendorf tube containing water and subsequently sent to Poland. The remaining nematodes were used to morphological and morphometric study, fixed with a hot 99:1 solution of 4% formaldehyde and pure glycerin (Seinhorst, 1962) and then processed to anhydrous glycerin (Seinhorst, 1959). Permanent slides were made and Tripylina nematode individuals were studied using a light microscope Eclipse 90i Nikon, Japan at 100, 200, 400, 600, and 1000 × magnification.

The soil samples collected in 2018 were extracted in Poland by the decantation and sieving method followed by the centrifugal flotation method (van Benzooijen, 2006). Extracted nematodes were heat-killed using tap water at 60°C. Some of the selected individuals (as well as a few individuals from 2016 that arrived alive in water from Slovakia) were fixed in TAF and subsequently submitted for morphological analysis. Photographs and measurements were taken from 16 females and 14 males with a Leica DFC 500 camera on a Leica DM 5000B microscope. The remaining nematodes were fixed in DESS and temporary slides were prepared. With photographic documentation collected, the examined specimens were washed with PBS and subsequently with sterile water and then submitted for molecular studies.

Body long and relatively slender, arcuate ventrally after gentle heat treatment with posterior part of body more curved than anterior. Cuticle smooth, 1.8 (1.4–2.3) µm thick. Body pores small and numerous, distributed along entire body. Pseudocoelomocyte cells 14.7 (10.2–20.5) µm wide and 27.2 (15.1–41.6) µm long, located along body, their number varying from one to seven pairs. Max. body diam. generally recorded at ovary level, occasionally at level of vulva. Head rounded, 29.3 (27.4–33.0) µm diam., smooth, continuous with body contour. Inner labial papillae conical, 2.2 (1.7–2.4) µm long. Six outer labial setae and four short cephalic setae arranged in a single whorl. Outer labial setae strongly developed, 21.2 (17.0–24.2) µm long, more or less arcuate, bent at tip and directed anteriorly, four cephalic setae 9.1 (7.1–12.4) µm long, thinner than outer labial setae. Stoma 31.6 (29.6–34.9) µm long, funnel-shaped with weakly sclerotised cheilostom, rest of stoma with dorsal wall thickened almost for its entire length, thickening gradually expanding from beginning of mouth to dorsal tooth, where wall thickness is largest. Dorsal tooth relatively large, triangular, directed posteriad, lying 28 (26–29.0) µm from anterior end of body. Two small subventral teeth 3.1 (2.3–3.7) µm anterior to dorsal tooth. Amphids cup-shaped, located 19.0 (17.4–20.6) µm from anterior extremity. Excretory pore 148 (133–175) µm from anterior end. Cervical region with three ventromedian setae. One female with two lateral setae lying 54 and 138 µm from anterior end of body. Pharynx cylindrical and muscular, slightly enlarged anteriorly to surround stoma. Dorsal pharyngeal gland opening at level of dorsal tooth. Nerve ring located at 39.6 (35.5–47.0)% of pharyngeal length from anterior end. Excretory pore not observed. Cardia located between pharynx and intestine, relatively large and wide with gland-like bodies. In intestine of several individuals an ingested nematode was observed. Rectum arcuate, 31.0 (26.8–34.2) µm long. Tail with poorly visible caudal glands, terminated by a small, tubular spinneret 2.1 (1.8–2.3) µm long. Two subdorsal caudal setae situated a short distance anterior and posterior to anus. Reproductive system 374 (224–726) µm long, monodelphic-prodelphic with post-vulval uterine sac 145 (103–190) µm long, occasionally containing large, oval sperm. Pars refringens vaginae clearly separated, tear-shaped, sometimes almost semi-spherical sclerotisations (4.8–6.6 µm long and 5.4–7.6 µm wide). Pars distalis vaginae clearly marked, thick walled. Vulva with protuberant lips.

Morphology and morphometrics similar to that of female. Genital branch 668 (312–849) µm long. Testis outstretched, filled with oval-shaped spermatozoa. Spicule narrow with arched proximal and acute distal end, 54 (48–59) µm long, evenly tapering, sickle-shaped. Gubernaculum well developed, 10.1 (9.0–11.8) µm long, bifurcate proximally, surrounding spicules dorsally and laterally. Cloaca 42.0 (34.0–49.2) µm long. Ventromedian, precloacal supplements five in number, papillate, irregularly arranged. Distance between first and last supplement 119 (77–159) µm. First supplement lying 8.3–11.9 µm from cloacal aperture, second 28–41, third 45–83, fourth 73–132, and fifth 96–167 µm.

Iran. Soil and litter, at a depth of 0–15 cm under a hawthorn tree (Crataegus monogyna L.) in a forest, Naharkhoran region, Gorgan, Golestan Province, northern Iran. One paratype female and two paratype males deposited at the National Nematode Collection, New Zealand (NNCNZ).

Slovak Republic. Soil and litter, at a depth of 0–20 cm under a beech tree (Fagus sylvatica L.) in a natural forest in Opátka (N 48°47’, E 21°04’, 776 m a.s.l.), a small village in the Košice district, eastern Slovakia. The description is based on the morphology and morphometry of 16 females and 14 males.

Voucher specimens were deposited in the National Nematode Collection in New Zealand (NNCNZ), the Crown Research Institute Landcare Research New Zealand Ltd, New Zealand (2 females, 2 males) and in the nematode collection at the U.S.D.A, Beltsville, USA (one female, one male). The remaining specimens were deposited in the Institute of Parasitology, Slovak Academy of Sciences, Slovak Republic (4 females, 4 males) and in the nematode collection at the Museum and Institute of Zoology, PAS, Warsaw, Poland (9 females, 7 males).

Specimens collected from a natural beech forest in Slovak Republic were first identified as an undescribed species, morphologically similar to Tripylina gorganensis described from Iran (Asghari et al., 2012). An important feature distinguishing both species was the presence of post-vulval uterine sac (PUS) in specimens from Slovakia, which, according to the original description (Asghari et al., 2012), was absent in Tripylina gorganensis. A careful re-examination of type specimens revealed that T. gorganensis also has a post-vulval uterine sac. As a consequence a redescription of T. gorganensis was needed. Morphologically, the specimens from Slovak Republic agree well with the paratypes of T. gorganensis. The morphometrics of the beech forest nematodes agrees with most of the dimensions in the original description except for some minor differences: they have a longer tail (76–95 vs 49–64 µm in females, 76–99 vs 45–67 µm in males) and a lower value of the coefficient c (18–25 vs 27–36 in females and 15–22 vs 24–40 in males). We consider these differences may be a part of the intraspecific variation within this species.

T. gorganensis is characterized by having a long body, dorsal wall thickened almost over the entire length of stoma, a large dorsal tooth directed posteriad, subventral denticles located anterior to dorsal tooth, three ventromedian setae and two pairs of lateral cervical setae in the cervical region, a relatively long post-vulval uterine sac, two subdorsal caudal setae situated a short distance anterior and posterior to anus and male having five to six papillate ventromedian supplements.

Tripylina gorganensis together with T. bravoae, T. iandrassyi and T. longa belongs to a small group of Tripylina whose common, distinctive features are: stoma with dorsal wall thickened almost on its entire length, the thickening gradually expands from the beginning of the mouth to the dorsal tooth, a large dorsal tooth directed posteriorly and the presence of males. Their close relationship, except T. longa for which ribosomal sequences are not available, is supported by the molecular data (Fig. 4). Phylogenetic analyses confirm the basal positioning of Tripylina gorganensis (Asghari et al., 2012) within a well supported Tripylina clade (Figs 4 and 5). BLAST analysis revealed the sequence identity between T. gorganensis populations from Iran and Slovakia of more than 99% (4 bp difference) in case of 18 S rDNA and of 97–98% (16–20 SNPs) in case of 28 rDNA.

Figure 4:

Figure 5:

Key to species of the Tripylina longa group