Research Article |
Corresponding author: Boris D. Efeykin ( bocha19@yandex.com ) Academic editor: Steven Nadler
© 2021 Elena S. Ivanova, Boris D. Efeykin, Sergei E. Spiridonov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ivanova ES, Efeykin BD, Spiridonov SE (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: 135-150. https://doi.org/10.3897/zookeys.1076.75932
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Synoecnema hirsutum Timm, 1959 (Ungellidae, Drilonematoidea), found in the body cavity of the pheretimoid earthworm at the border of Laos and Vietnam, was re-described and illustrated. The mitochondrial genome of S. hirsutum obtained with Illumina HiSeq sequencing is the first annotated mitochondrial genome as a representative of the superfamily Drilonematoidea. The phylogeny inferred from the analysis of 12 mitochondrial genes has shown some similarity of S. hirsutum with a cephalobid Acrobeloides varius.
Drilonematids, ungellids, earthworms, Vietnam, mitochondrial genome, phylogeny
Recent phylogenetic analyses have shown that multiple (18 according to Viney 2017) separate occasions of transition to parasitic lifestyle had occurred in the evolutionary history of nematodes. Phylogenetic relationships of parasitic nematodes and free-living representatives of this phylum can reveal the evolutionary pathways of the acquisition of parasitism in this group of animals. However, the evolutionary history of the most numerous group of parasitic nematodes – the order Rhabditida (formerly the subclass or the class Secernentea) still remains a puzzle. The analysis of contemporary literature demonstrates that different groups of parasites have been studied in a very inconsistent manner with the bias on the parasitic nematodes of humans, domesticated animals and plants versus the parasitic nematodes of economically-unimportant invertebrates. The latter are also poorly presented in the mitochondrial genome phylogenetic research.
It is evident that evolutionary processes of parasitism acquisition are not anchored to the organisms important for humans and the study of neglected groups of parasites and their hosts can reveal interesting evolutionary patterns. One of such groups is the Drilonematoidea, one of the larger nematode taxa with still unresolved phylogeny. It hosts the highly specialised and diverse group of coelomic parasites of earthworms (Annelida, Clitellata). The impressive diversity of the nematodes, parasitic in earthworms, was discovered and described by R.W.
Within Drilonematoidea, the family Ungellidae (ungellids) is the most speciose taxon with the morphology profoundly changed by the parasitic lifestyle. These nematodes are characterised by the presence of cephalic hooks, expanded glandular structures in the enlarged tail portion of the body, degeneration of a spicular apparatus in males and thick-walled eggshells. They are very rare in earthworms inhabiting temperate regions, but found in the earthworm taxa of tropics and subtropics. Such geographical distribution of ungellids is a serious obstacle to the examination of this group with modern techniques.
The species of Synoecnema (Ungellidae) was found by S. E. Spiridonov in the body cavity of a pheretimoid earthworm collected in Vietnam at the border with Laos in April 2019. The genus Synoecnema had been established by de Magalhães in 1905 to accommodate a nematode species (S. fragile) found in the body cavity of an earthworm and characterised by the presence of a pair of cephalic hooks. Its taxonomic status was revised by
So far, all species of Synoecnema were found in coelomic cavities of tropical earthworms belonging to Megascolecoidae (mainly) and Drawidae and collected in South America, Papua New Guinea, India and Southeast Asia. To date, the genus Synoecnema Magalhães, 1905 accounts for 20 species: S. fragile Magalhães, 1905, S. acutifrons Pierantoni, 1916, S. anseriforme Timm, 1959, S. (= Siconemella) burmensis Timm, 1967, S. drawidae Baylis, 1943, S. gatesi Timm, 1962, S. guinensis Pierantoni, 1916, S. hirsutum Timm, 1959, S. hoplochaetellae Baylis, 1943, S. laotense Spiridonov, 1993, S. modigliani Ivanova & Spiridonov, 1989, S. perionychis Baylis, 1943, S. pheretimae Baylis, 1943, S. (= Siconemella) philippinensis Timm, 1967, S. pingi Ivanova & Spiridonov, 1989, S. robustum Ivanova & Spiridonov, 1989, S. rodericensis Ivanova & Spiridonov, 1989, S. tsiliensis Ivanova & Spiridonov, 1989, S. tuliemense Ivanova and Pham Van Luc, 1989 and S. watinagii Ivanova, Sumaya & Spiridonov, 2015.
To date, the latter species is the only member of the genus molecularly characterised. It is suggested that new discoveries in the morphology and genetics of the members of the genus may bring the need for further revision. The quantity of material collected allowed us to obtain enough material to provide the extended molecular analysis aimed at the resolution of relationships between higher taxa of Drilonematoidea, parasites of earthworms.
Nematode material. Earthworms were collected at Muóng Lát, Thanh Hóa Province, VietNam (20°31'N, 104°56'E) in April 2019 and dissected alive. Fourteen specimens of the earthworm host species were examined and all were found infected by 2–80 nematodes (av. 14). All nematodes were presented by adult stages with the majority found in a state of a permanent copula, characteristic to the genus. Nematodes were located in the body cavity along the whole host body with the majority occupying anterior segments, were not attached to the septae, but were nearly immobile and responded to the dissection of the host by slight twitching soon followed by its distortion, bursting and death. For the morphological examination, about 20 specimens were fixed by hot 4% formalin and the rest by 96% ethanol for molecular studies.
Nematodes, preserved in formalin, were processed by anhydrous glycerine for light microscopy as described by
DNA from the frozen nematode samples was isolated using the QiAmp Micro Kit (Qiagen) according to a standard protocol. DNA library preparation was implemented using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The DNA quality was checked with Qubit 3.0, final library length distribution and checking for the absence of adapters was performed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing was performed on Illumina HiSeq 4000 system with a 150 bp read length at the Skoltech Genomics Core Facility (https://www.skoltech.ru/research/en/shared-resources/gcf-2/).
The quality of raw reads was evaluated using FastQC (
Phylogenetic reconstructions were conducted from the alignment of 12 protein-coding genes with Limulus polyphemus and Lithobius forficatus as outgroups. For multiple alignments of AA sequences, the nucleotide sequences of each of the protein-coding genes were initially translated into AA with MEGA6. Conserved regions in the alignments of the 12 PCGs were selected using the GUIDANCE2 server (
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.
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
For S. anseriforme females,
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 (
The results of the multigene phylogenetic analysis of Synoecnema hirsutum relationships are presented in Fig.
The circular molecule of complete mitochondrial genome has been reconstructed from separate contigs (Fig.
The superfamily Drilonematoidea is a taxon representing a quite exotic and not easily obtainable group of parasites. Due to little or no molecular data available for the majority of their taxa, relationships within four families constituting Drilonematoidea are still not phylogenetically resolved. Likewise, it is true concerning two subfamilies of Ungellidae (Ungellinae and Synoecneminae) where the present species belongs. The family Ungellidae has been split into Synoecneminae accommodating species lacking male spicular apparatus and Ungellinae housing ones with spicules and a gubernaculum (
Limited molecular data are available for some of the other higher taxa of Drilonematoidea and include several SSU and LSU sequences for two representatives of Homungellidae and representatives of three genera of Drilonematidae. The previous phylogenetic analyses were not in full agreement with the classification, based on morphology and topologies, showing the different positions on the phylogenetic tree for parasites from lumbricid and non-lumbricid earthworm hosts. The ‘lumbricid’ parasites were presented by Dicelis species from the Drilonematidae family (D. lovatiana Ivanova, 1993; D. kimmeriensis Ivanova, 1993; D. rubidi Ivanova, 1994; D. caledoniensis Spiridonov, Ivanova & Wilson, 2005; D. ussuriensis Spiridonov, Ivanova & Wilson, 2005) and were separated from the rest of Drilonematoidea phylogenetically representing a terminal branch of cephalobid phylogeny (
In the present study, we aimed to overcome the limitations of the previous molecular phylogenetic analyses using a multigene analysis, based on 12 protein-encoding genes. Earlier, it has been shown that some mitochondrial genes are not suitable for the analysis of phyletic links between higher taxa because of saturation (Blouin et al. 1998). Still, we expected the wider set of genes to bring sufficient discriminatory power to reveal the phylogenetic relationships within Drilonematoidea. The close position of the ungellid Synoecnema hirsutum to the cephalobid Acrobeloides varius in the present analysis corresponds to the previous topologies (
Previous attempts to find the place for ungellids and other Drilonematoidea in the system of Nematoda were based on nuclear ribosomal sequences only (
The assembled mitochondrial genome is deposited in NCBI GenBank under accession MG294556. Protocols are deposited in protocols.io under: https://doi.org/10.17504/protocols.io.bv4gn8tw
We gratefully acknowledge the financial support from the Russian Science Foundation grant [19-74-20147].