Synoecnema hirsutum Timm, 1959

Ivanova, Elena S., Efeykin, Boris D. & Spiridonov, Sergei E., 2021, The re-description of Synoecnema hirsutum Timm, 1959 (Synoecneminae, Ungellidae, Drilonematoidea) from a pheretimoid earthworm in Vietnam with the analysis of its phylogenetic relationships, ZooKeys 1076, pp. 135-150 : 135

Synoecnema hirsutum Timm, 1959

General.

Adults. Small nematodes lacking sexual dimorphism in body shape and of anterior end structure. Body almost cylindrical, tapering to both ends. Cuticle thin, transversely striated, bearing diverse short setae and spike-like outgrowths variously distributed in different specimens. Epidermis thick. Lateral fields absent. Anterior end curved bearing small paired cephalic hooks. Hooks sub-terminal, curved, thickened and directed dorsad. Hook base ca. 2 µm long, hook blades ca. 3 µm long, closely positioned with distal tips slightly diverging and directed towards or parallel to hook base. Cephalic sensilla indistinct. Amphids discernible in several specimens; apertures situated closely to hook base, elliptical, 1-2 µm wide. Mouth shifted ventrad; stoma absent. Pharynx clavate with long thin muscular corpus and large muscular-glandular pear-shaped terminal bulb displaced dorsally. Isthmus not expressed. Nerve ring encircling posterior of corpus. Excretory pore 1 µm wide, level with nerve ring, excretory duct strongly cuticularised, extending beyond bulb base, paired excretory canals weakly cuticularised, passing through very large excretory gland which can be tracked at least to mid-body. Intestine discernible at anterior, cardia-like structure present. Caudal organs long shallow grooves situated mid-laterally on the surface of posterior half of body; grooves’ surface lacking cuticle. No duct inside caudal organ observed. Tail extremity conical. Sexes often permanently in copula.

Female: N = 9. Body length = 1185 ± 138 (1014-1385) µm; a = 22.2 ± 3 (19-28); b = 11.2 ± 1.5 (9-13.2); max width = 64 ± 7 (55-75) µm; pharynx length = 107 ± 10 (93-123) µm; basal bulb height = 34 ± 3 (30-38) µm; basal bulb width = 19 ± 2 (16-22) µm; nerve ring from apex = 61 ± 10 (50-75) µm; excretory pore from apex = 74 ± 15 (54-101) µm; spermatheca from apex = 139 ± 18 (120-176) µm; V% = 44.5 ± 0 (36.1-49.5); egg length = 47 ± 2 (43-49) µm; egg width = 20 ± 1 (18-22) µm.

Anterior end tapering from pharyngeal base level. Pharyngeal procorpus 4-5 µm wide. Prodelphic, monodelphic. Postvulval body region very slightly swollen. Multilobed gland of obscure function present at posterior portion of body behind vulva, in some specimens hindering observation of gonad track and entwining gonad branches. Ovary distal cell situated close to tail extremity. Ovary running anteriad to the level of postvulval region, then turning posteriad to the point of distal cell and then turning again anteriad where it runs straight ahead until reflexing at some distance (about corresponding body diameter) behind pharynx base. At reflexion, gonad forming large, not distinctly offset spermatheca (av. size 56 µm × 34 µm), followed by thick-walled oviduct and spacious thin-walled uterus. Spermatheca filled with large spermatozoa ca. 2 µm in diameter. Vulva pre-equatorial, on slight protuberance, anterior vulval flap enlarged, vagina absent and vulva opens immediately into uterus. No post-uterine sack present. Eggs ovoid, arranged in a single row, 3-5 with fully-developed eggshells at a time. Fully-developed eggshells 1 µm thick densely covered with spikes 2 µm long. Tail pointing posterior to ovary distal cell; portion of tail free of gonad short. Rectum and anus indiscernible. Caudal organs extending from vulva level to nearly end of tail.

Male: N = 5. Length = 624 ± 77 (517-725) µm; a = 18.5 ± 0.7 (17.8-19.1); b = 5.3 ± 1.1 (3.9-6.1); c = 2.8 ± 9.5 (2.2-3.1); c’ = 7.9 ± 2.7 (6-10.9); max width = 36 ± 3 (32-38) µm; pharynx length = 129 ± 25 (103-167) µm; basal bulb height = 36 ± 3 (32-40) µm; basal bulb width = 18 ± 2 (15-20) µm; nerve ring from apex = 73 ± 1 (72-74) µm; excretory pore from apex = 73 ± 11 (60-82) µm; testis reflexion from apex = 316 ± 102 (260-497) µm; testis reflexion length = 64 ± 18 (40-83) µm; tail length = 240 ± 51 (201-328) µm.

Very similar to females in general appearance and morphology of anterior end and caudal organs, but much smaller and slimmer, especially at posterior. Monorchic. Testis reflexed at anterior third of body level. Flexure short and wide. Spermatocytes rounded, ca. 5 µm in diameter, arranged distally in two rows. Proximal part of reproductive system not distinctly differentiated into vas deferens and ejaculatory duct. Spicular apparatus and gubernaculum absent. Anal flaps developed unequally, anterior flap inflated and hook-like, while posterior one much smaller, partly overhanging indentation posterior to anus. No caudal sensilla detected. Caudal organ structure and position similar to that of females.

Remarks on morphology

The examination of the present species has shown its strong similarity to S. hirsutum Timm, 1959 in the general morphology, i.e. body proportions, the shape and size of cephalic hooks and eggs and the cuticle appearance. In terms of morphometrics, there are a few smaller differences which include: the slightly larger body size (1014-1385 µm vs. 0.70-1.09 mm, females and 517-725 µm vs. 512-654, males), the longer male tail (201-328 µm vs. 160-244 µm) and the slightly more posterior vulva position (36.1-49.5 vs. 34.8-46.5%) (see Table 1). Contrary to Timm (1959), we did not observe an anus in any female specimen. Therefore, we concluded it was absent or indistinct. Describing S. anseriforme and S. hirsutum , Timm (1959) discovered in females a median ventral pore located posterior to a vulva. The pore was interpreted by the author as an additional excretory pore due to the absence of a distinct intestine and rectum in these nematodes. Later, Timm (1966) re-appraises such a ventral pore of S. anseriforme as an anal aperture, but does not mention S. hirsutum . He also notes that an anal aperture is absent in the rest of the Synoecnema .

For S. anseriforme females, Timm (1959) also describes pocket-like phasmids or “suckers” and depicts similar structures for a female of S. hirsutum , but after re-examination of all known Synoecnema species of Baylis (1934) and Timm (1959, 1962), comes to the conclusion that "all have small cephalic hooks and lack distinct suckers in both sexes, with the exception of S. drawidae Baylis, 1934 in which the female has long narrow suckers with a slit-like opening". In many illustrations to the species of Synoecnema , both of Baylis and Timm, the long narrow slits were depicted (though not explained) extending along the posterior body portion of a nematode. Further on, Ivanova and Spiridonov (1987), Ivanova and Pham Van Luc (1989) and Spiridonov (1993) described similar structures in Synoecnema species using light microscopy as long, slit-like caudal organs surrounded by a modified (fibrous) tissue. Structure of similar slits/grooves in S. watinagii and an undescribed Synoecnema from pheretimoid hosts was examined with the aid of scanning microscopy (Ivanova et al. 2017; unpubl.). The slits were deep (immersed in body wall) or shallow, all located mid-laterally and extended from mid-body to near end of the tail. The surface of a slit was devoid of a cuticle. No sucker-like structures were observed.

As the chief diagnostic feature of S. hirsutum seems to be the presence of setae covering the body of the nematode which also is a characteristic trait of the Synoecnema from our material, we tend to assume that both nematodes belong to the same species.

The term "caudal organ" was used when describing caudal structures of yet unknown function in nematodes of Drilonematoidea ( Spiridonov et al. 2007; Ivanova and Neuhaus 2009; Ivanova and Bain 2013). Ivanova et al. (2017) discussed its possible function and pointed out the uniqueness of this structure in Synoecnema along with the simplifying of many other morphological traits.

Multigene analysis of Synoecnema hirsutum phylogenetic relationships

The results of the multigene phylogenetic analysis of Synoecnema hirsutum relationships are presented in Fig. 4 . The monophyly of Chromadoria nematodes, included in this analysis, is strongly supported as also the monophyly of the phylum Nematoda (Fig. 4 ). In this ML phylogenetic tree, S. hirsutum formed a clade under maximal bootstrap support with a soil free-living nematode Acrobeloides varius ( Kim et al. 2020). As the mitochondrial data for the only other acrobelid studied, A. complexus (KM192361), are incomplete, they were not used in the analysis. The clade Synoecnema + Acrobeloides clustered with tylenchids under maximal bootstrap support. Contrary to expectations, the combined clade of tylenchids + Synoecnema + Acrobeloides remained outside of the larger clade containing all other Rhabditida (sensu De Ley and Blaxter 2002) and the two studied Plectidae ( Plectus aquatilis and P. acuminatus ). Additionally, a clade containing oxyurids and spirurids was in a similarly detached position. Such topology with the basal position of the clade (tylenchids + Synoecnema + Acrobeloides ) was also observed in the trees inferred from the analysis of single mitochondrial genes atp 6, cox I, co b, nad 3, nad 5 and nad 6 (data not shown). The phylograms inferred from analyses of other mitochondrial genes (cox 2, cox 3, nad 1 nad 2, nad 4 and nad 4 L) demonstrated more traditional basal position of plectids vs. all other representatives of Rhabditida . Coalescent analysis (not shown) demonstrated the great similarity with the ML tree (Fig. 4 ).

An arrangement of the genes in the mitochondrial genome of Synoecnema hirsutum

The circular molecule of complete mitochondrial genome has been reconstructed from separate contigs (Fig. 3 ). With the length of 15396 bp, this circular genome contains 12 protein-encoding genes, 22 transport RNA genes, two ribosomal sequences and three non-coding regions.

Figures

Figure 3. Map of the mitochondrial genome of Synoecnema hirsutum. Non-coding areas are shaded.

Figure 4. Phylogenetic relationships of Synoecnema hirsutum inferred from Maximum Likelihood analysis of concatenated dataset of 12 mitochondrial protein-coding genes.