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All issues Volume 32 (2025) Parasite, 32 (2025) 14 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 14
Number of page(s) 14
DOI https://doi.org/10.1051/parasite/2025008
Published online 24 February 2025

© X. Gu et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Species of Seuratascaris Sprent, 1985 (Ascaridida: Ascaridoidea) are a rarely reported group of ascaridoid nematodes, specifically parasitizing various frogs and toads [3, 4, 17, 24]. Sprent (1985) [24] erected the genus Seuratascaris. Later, Fagerholm (1991) [5] assigned this genus to the Angusticaecinae Skrjabin & Karokhin, 1945 in the Ascarididae Baird, 1853. To date, only four nominal species have been reported from Europe, Oceania and South East Asia, namely S. numidica (Seurat, 1917), S. ranae (Wang, Zhao & Chen, 1978), S. physalis Chen & Li, 2023 and S. schmackeri Liu, Fang, Zheng & Wu, 2023 [3, 4, 17, 24, 25]. Although the recent studies sequenced some nuclear and mitochondrial data of this group [3, 4], there has been no mitochondrial genomic data for species of Seuratascaris reported.

In the present study, some specimens of Seuratascaris collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China, were identified to species level using integrated morphological methods (light and scanning electron microscopy) and molecular approaches [sequencing the nuclear internal transcribed spacer (ITS) regions and mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and small subunit ribosomal RNA gene (rrnS)]. The Assemble Species by Automatic Partitioning (ASAP) analyses and Bayesian inference (BI) based on the ITS, cox1, cox2 and rrnS sequence data, were also used for species delimitation of Seuratascaris spp., respectively. In order to further reveal the characterization of mitochondrial genome of ascaridoid nematodes, the mitogenome of the present material was also sequenced and annotated, which represents the first mitogenomic data for the genus Seuratascaris.

Materials and methods

Ethics

This study was conducted under the protocol of Hebei Normal University (LLSC2024090). All applicable national and international guidelines for the protection and use of animals were followed.

Morphological observation

A total of 21 individuals of Odorrana graminea were caught by hand at night in Bazhaigou Scenic Area, Qinzhou city, Guangxi Zhuang Autonomous Region, China, and euthanized by the double marrow destruction method [12]. Nematode specimens were collected from the digestive tract. Parasites were washed in physiological saline, then stored in 80% ethanol until studied. The morphology of several specimens of S. phy. physalis newly collected from Odorrana tiannanensis (Yang & Li) (Anura: Ranidae) and Rhacophorus sp. (Anura: Rhacophoridae) in Yunnan Province, China, were also studied.

For light microscopic study, nematodes were cleared in lactophenol. Photomicrographs were recorded using a Nikon® digital camera coupled to a Nikon® optical microscope (Nikon ECLIPSE Ni-U, Nikon Corporation, Tokyo, Japan). For scanning electron microscopy (SEM), the cephalic extremity and posterior end of one male and one female were re-fixed in 4% formaldehyde solution, post-fixed in 1% OsO4, dehydrated via an ethanol series and acetone, and then critical point dried. Samples were coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 kV. Measurements are given in millimetres (mm). Nematode specimens were assigned to the genus Seuratascaris based on the following features according to previous studies [3, 4, 24], including lips possessing dentigerous ridges, the absence of interlabia, ventriculus, ventricular appendage and gubernaculum, the excretory pore just posterior to the nerve ring, the presence of an intestinal caecum and spicules very short without alae.

Molecular procedures

The mid-body of 2 nematode individuals (1 male and 1 female) collected from O. graminea in Guangxi, and 3 individuals of S. phy. physalis (1 male and 1 female from Rhacophorus sp., and 1 female from O. tiannanensis) were used for molecular analysis. Genomic DNA was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, Shanghai, China), according to the manufacturer’s instructions. DNA was eluted in elution buffer and kept at −20 °C until use. The used primers and cycling conditions for amplifying the target sequences of ITS, cox1, cox2 and rrnS, and the procedures for sequencing and analysing were according to previous studies [3, 10]. All of the sequences obtained herein were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) under the following accession numbers: the present new subspecies: ITS: PQ535527, PQ535528; cox1: PQ535571, PQ535572; cox2: PQ554528, PQ554529; rrnS: PQ535525, PQ535526. S. phy. physalis: ITS: PQ818925–PQ818927; cox1: PQ817697–PQ817699; cox2: PQ819747–PQ819749; rrnS: PQ818928–PQ818930.

ASAP and BI analyses

The ASAP analyses were executed using the ASAP online server (https://bioinfo.mnhn.fr/abi/public/asap) under the Kimura (K80) ts/tv model based on the ITS, cox1, cox2 and rrnS sequence data, respectively. The results of ASAP with lowest scores were considered as the optimal group number in the present study. Bayesian inference was performed using MrBayes 3.2.7 with two parallel runs (1,000,000 generations) under the following optimal models (i.e., HKY + G model for cox1, HKY + G model for cox2, HKY + G model for rrnS, and DAYHOFF + I model for ITS). Ophidascaris baylisi (Robinson) (Ascaridoidea: Ascarididae) was chosen as the out-group for both ASAP and BI analyses.

Mitochondrial genome sequencing, assembly and annotation

A total of 35 Gb clean genomic data were generated using the Pair-End 150 sequencing method on the Illumina NovaSeq 6000 platform by Novogene (Tianjin, China). The complete mitochondrial genomes were assembled using GetOrganelle v1.7.2a [14]. Protein coding genes (PCGs), rRNAs and tRNAs were annotated using MitoS web server (http://mitos2.bioinf.uni-leipzig.de/index.py) and MitoZ v3.6 [18]. The open reading frame (ORF) of each PCG was confirmed manually by the web version of ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The “lost” tRNA genes ignored by both MitoS and MitoZ, were identified using BLAST based on a database of the existing tRNA sequences of nematodes. The secondary structures of tRNAs were predicted by ViennaRNA module [8], building on MitoS2 [2] and RNAstructure v6.3 [22], followed by manual correction. The base composition, amino acid usage and relative synonymous codon usage (RSCU) were calculated by Python script, which refers to the Codon Adaptation Index (CAI) [15]. The total length of the base composition included ambiguous bases. The base skew analysis was used to describe the base composition of nucleotide sequences. The complete mitochondrial genome of the present material obtained herein was deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) under accession number PQ468471.

Results

Seuratascaris physalis bazhaiensis n. subsp. (Figs. 1–4)

urn:lsid:zoobank.org:act:9B09A6D3-8E5A-46BB-81EE-70A549A0A951

thumbnail Figure 1

Scanning electron micrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China, male. A: anterior part of body (excretory pore arrowed), lateral view; B: magnified image of excretory pore, ventral view; C: magnified image of excretory pore, lateral view; D: dorsal lip; E: subventral lip; F: tail (postcloacal papillae indicated using white arrows, phasmid indicated using black arrow), ventral view; G: magnified image of precloacal papillae; H: magnified image of medio-ventral precloacal papilla; I: magnified image of postcloacal double papillae; J: magnified image of postcloacal papillae; K: posterior end of body (precloacal papillae arrowed), ventral view; L: magnified image of tail tip. Abbreviations: am, amphid; dp, double papilla; sp., single papilla.

Type host: Odorrana graminea (Boulenger, 1899) (Anura: Ranidae).

Type locality: Bazhaigou Scenic Area, Guangxi Zhuang Autonomous Region, China.

Site of infection: Intestine.

Level of infection: 14.3% (3 out of 21 individuals of O. graminea) were infected with intensity of 2.0–4.0 (3.0) nematodes.

Type specimens: Holotype: male (HBNU–N–A20240920GL); allotype: female (HBNU–N–A20240921GL); paratypes: 1 male, 4 females (HBNU–N–A20240922GL), deposited in the College of Life Sciences, Hebei Normal University, Hebei Province, China. Paratypes: 2 females (NZMC–PN_506–507), deposited in the National Zoological Museum, Beijing, China.

Etymology: The subspecies name refers to the type locality (Bazhaigou Scenic Area).

Morphology

Small to medium sized, whitish nematodes. Cuticle with fine, transverse striations. Maximum width of body at about mid-body. Cephalic extremity with 3 lips, almost equal in size (Figs. 1A, 2A, 2B, 3A–3C, 4A–4C). Dorsal lip with 1 pair of large double cephalic papillae (Figs. 1D, 3B, 4B); subventral lips each with single double cephalic papilla, small papilla and amphid (Figs. 1E, 3C). Distal margin of each lip armed with 75–85 conical denticles and single small triangular, medio-apical notch (Figs. 1D, 1E, 2A–2C). Interlabia absent (Figs. 1A, 2A, 2B, 4C). Cuticle in cervical region inflated to form a cephalic vesicle-like structure extending posteriorly to anterior 1/4 of intestinal caecum (Figs. 3A, 4A). Oesophagus muscular, narrow, nearly cylindrical (Figs. 3A, 4A). Ventriculus and ventricular appendix absent. Intestinal caecum long, about 2/3 of oesophageal length (Figs. 3A, 4A). Nerve-ring at about 1/6 of oesophageal length. Excretory pore just posterior to nerve-ring (Figs. 3A, 4A). Cervical papillae not observed. Cuticle in posterior end of male and female both inflated (Figs. 3E, 3G, 4E, 4G). Tail of both sexes conical, with very small digitiform tip (tail tip retracted, nearly not observed in some individuals under LM) (Figs. 1F, 1K, 1L, 2D, 2E, 3E, 3G, 3I, 4E–4G).

thumbnail Figure 2

Scanning electron micrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China, female. A: cephalic end, apical view; B: cephalic end, lateral view; C: magnified image of denticles; D: magnified image of tail tip (phasmids arrowed); E: posterior end of body, ventral view; F: region of vulva, ventral view.

thumbnail Figure 3

Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China. A: anterior part of male body, lateral view; B: cephalic end of male, dorsal view; C: cephalic end of male, sublateral view; D: region of vulva, lateral view; E: tail of female, lateral view; F: egg; G: posterior end of male, ventral view; H: spicules; I: posterior end of male, lateral view.

thumbnail Figure 4

Photomicrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China. A: anterior part of male, lateral view; B: cephalic end of male, dorsal view; C: cephalic end of male, ventral view; D: region of vulva, lateral view; E: posterior end of female, lateral view; F: posterior end of male, lateral view; G: posterior end of male, ventral view.

Male (Based on 2 mature specimens): Body 10.4–11.5 (10.9) long; maximum width 0.33–0.37 (0.35). Dorsal lip 0.087–0.11 (0.097) long, 0.067 wide. Oesophagus 2.44–2.79 (2.62) mm long, 0.082–0.097 (0.089) in maximum width, representing 23.5–24.3 (23.9)% of body length. Intestinal caecum 1.82–1.84 (1.83) mm long, 0.08–0.11 (0.095) wide, representing 65.9–74.6 (70.3)% of oesophageal length. Nerve-ring 0.42–0.47 (0.45) and excretory pore 0.43–0.47 (0.45) from cephalic extremity, respectively. Posterior end of body distinctly curved ventrally (Figs. 1K, 3I, 4F). Spicules robust, short, without alae, equal in length, blunt at distal end, 0.16–0.17 (0.16) long, representing 1.47–1.49 (1.48)% of body length (Figs. 3H, 3I, 4F, 4G). Gubernaculum absent. Caudal papillae 8–10 pairs in total, arranged as following: 4–6 pairs precloacal (number and/or arrangement of precloacal papillae distinctly asymmetrically on two sides) and 3 pairs postcloacal (first pair being double papillae) (Figs. 1F–1K, 3G, 3I, 4F, 4G). Single medio-ventral precloacal papillae located at some distance anterior to precloacal lip (Figs. 1K, 3G). Precloacal or postcloacal ornamentation absent. Tail 0.25–0.28 (0.27) long. Phasmids small, located laterally, at base of tail tip (Fig. 1F).

Female (Based on 7 gravid specimens): Body 27.9–58.2 (46.6) long; maximum width 0.56–1.27 (0.84). Dorsal lip 0.16–0.22 (0.20) long, 0.11–0.18 (0.14) wide. Oesophagus 3.68–7.83 (5.44) long, 0.14–0.20 (0.17) in maximum width, representing 9.92–14.4 (11.8)% of body length. Intestinal caecum 2.59–4.76 (3.16) long, 0.15–0.24 (0.19) wide, representing 49.4–79.5 (58.8)% of oesophageal length. Nerve-ring 0.63–0.92 (0.81) and excretory pore 0.67–0.97 (0.87) from cephalic extremity, respectively. Vulva slit-like, vulval flap absent, 9.41–22.1 (15.6) mm from cephalic extremity, representing 29.7–40.7 (33.4)% of body length (Figs. 2F, 3D, 4D). Vagina muscular, very short; uterus didelphic. Eggs oval, with finely pitted shell, 0.10–0.15 (0.13) × 0.08–0.11 (0.09) in size (n = 30) (Figs. 3F, 4D). Tail 0.25–0.44 (0.34) long. Phasmids small, located laterally, at base of tail tip (Figs. 2D, 3E, 4E).

Remarks

In the genus Seuratascaris, S. phy. bazhaiensis n. subsp., together with S. phy. physalis, can be easily distinguished from S. ranae and S. numidica by the presence of the distinctly inflated cuticle forming a cephalic vesicle-like structure at the anterior part of the body (vs. the absence of an inflated cuticle forming a cephalic vesicle-like structure at the anterior part of the body in the latter two species). Additionally, the new subspecies is distinguished by a relatively longer oesophagus in the male (oesophageal length representing 23.5–24.3% of the body length in S. phy. bazhaiensis vs representing about 11.0–20.6% of body length in S. ranae and S. numidica) [4, 24, 25].

Seuratascaris physalis physalis was originally described based only on one male and one female specimen [3]. In the present study, supplementary morphometric data of S. phy. physalis were provided based on newly collected specimens from O. tiannanensis and Rhacophorus sp. in Yunnan Province, China (see Table 1 for details), which can contribute to better understanding of the ranges of intraspecific morphological variation and enable us to diagnose this subspecies more accurately. Chen & Li (2023) [3] reported the cephalic vesicle-like structure extending posteriorly to the level of excretory pore in S. phy. physalis based on the limited material. However, in the newly collected specimens of S. phy. physalis, we found that the cephalic vesicle-like structure can also extend from the base of the cephalic end nearly to the apex of the intestinal caecum in some individuals. Consequently, in spite of the cephalic vesicle-like structure extending posteriorly to the anterior 1/4 of the intestinal caecum in S. phy. bazhaiensis, it is not easy to distinguish this new subspecies from S. phy. physalis based on this feature. Although the newly collected male specimens of S. phy. physalis also exhibited broad ranges of morphological variability in the lengths of the body and oesophagus, the characteristics of tail length, and the ratio of oesophageal length to body length in the males of S. phy. physalis seem to be stable (S. phy. physalis with tail 0.10–0.23 mm and oesophageal length representing 14.5–17.9% of body length in males), which are different from that of S. phy. bazhaiensis (tail 0.25–0.28 mm and oesophageal length representing 23.5–24.3% of body length in males).

Table 1

Morphometric comparisons of Seuratascaris physalis bazhaiensis and Seuratascaris physalis physalis. Measurements are given in micrometers (μm).

Key to species/subspecies of Seuratascaris Sprent, 1985

1. Cuticle of the cervical region not inflated to form a cephalic vesicle-like structure...........2.

Cuticle of the cervical region distinctly inflated to form a cephalic vesicle-like structure...........3.

2. Male with 12 pairs of precloacal papillae, vulva located at 56.0% of body length...........S. ranae.

Male with no more than 9 pairs of precloacal papillae, vulva located at 24.7–41.2% of body length...........S. numidica.

3. Tail 0.16–0.23 mm long and oesophageal length representing 14.5–17.9% of body length in male...........S. physalis physalis.

Tail 0.25–0.28 mm long and oesophageal length representing 23.5–24.3% of body length in male...........S. physalis bazhaiensis n. subsp.

Genetic characterization

Partial ITS region

Two ITS sequences of S. phy. bazhaiensis obtained herein are both 974 bp in length, with no nucleotide polymorphism detected. In the genus Seuratascaris, the ITS sequence data are available in GenBank for S. numidica (MG434689, MG434690) and S. phy. physalis (OP330325). Pairwise comparison of the ITS sequences of S. phy. bazhaiensis with that of S. numidica and S. phy. physalis available in GenBank displayed 0% (S. phy. physalis) to 5.79% (S. numidica) nucleotide divergence. Three ITS sequences of S. phy. physalis (PQ818925–PQ818927) obtained herein are all 945 bp in length, with no nucleotide polymorphism detected. Pairwise comparison of the newly sequenced ITS data of S. phy. physalis with that of S. phy. bazhaiensis obtained herein, and S. phy. physalis (OP330325) and S. numidica (MG434689, MG434690) available in GenBank displayed 0%, 0%, and 5.55% nucleotide divergence, respectively.

Partial cox1 region

Two cox1 sequences of S. phy. bazhaiensis obtained herein are both 384 bp in length, with no nucleotide polymorphism detected. In the genus Seuratascaris, the cox1 sequence data are available in GenBank for S. numidica (MG434691, MG434692) and S. phy. physalis (OP329215). Pairwise comparison of the cox1 sequences of S. phy. bazhaiensis with that of S. numidica and S. phy. physalis available in GenBank displayed 5.73% (S. phy. physalis) to 11.2% (S. numidica) nucleotide divergence. Three cox1 sequences of S. phy. physalis (PQ817697–PQ817699) obtained herein are all 384 bp in length, representing two different genotypes, which exhibited 0–0.26% nucleotide divergence. Pairwise comparison of the newly sequenced cox1 data of S. phy. physalis (PQ817697–PQ817699) with that of S. phy. bazhaiensis obtained herein, and S. phy. physalis (OP329215) and S. numidica (MG434691, MG434692) available in GenBank displayed 5.99–6.25%, 1.82–2.08%, and 12.5–12.8% nucleotide divergence, respectively.

Partial cox2 region

Two cox2 sequences of S. phy. bazhaiensis obtained herein are both 501 bp in length, with no nucleotide polymorphism detected. In the genus Seuratascaris, the cox2 sequence data are available in GenBank for S. numidica (OP354281, OP354282) and S. phy. physalis (OP354280). Pairwise comparison of the cox2 sequences of S. phy. bazhaiensis with that of S. numidica and S. phy. physalis available in GenBank displayed 8.18% (S. phy. physalis) to 12.2% (S. numidica) nucleotide divergence. Three cox2 sequences of S. phy. physalis (PQ819747–PQ819749) obtained herein are all 501 bp in length, representing two different genotypes, which exhibited 0–0.60% nucleotide divergence. Pairwise comparison of the newly sequenced cox2 data of S. phy. physalis (PQ819747–PQ819749) with that of S. phy. bazhaiensis obtained herein, and S. phy. physalis (OP354280) and S. numidica (OP354281, OP354282) available in GenBank displayed 8.18–8.58%, 1.86–2.09%, and 11.1–11.2% nucleotide divergence, respectively.

Partial rrnS region

Two rrnS sequences of S. phy. bazhaiensis obtained herein are both 464 bp in length, with no nucleotide polymorphism detected. In the genus Seuratascaris, the rrnS sequence data are available in GenBank for S. numidica (OP331197, OP331198) and S. phy. physalis (OP330326). Pairwise comparison of the rrnS sequences of S. phy. bazhaiensis with that of S. numidica and S. phy. physalis available in GenBank displayed 4.51% (S. phy. physalis) to 8.42% (S. numidica) nucleotide divergence. In the present study, we also sequenced the rrnS sequences of S. phy. physalis based on newly collected specimens from Rhacophorus sp. and O. tiannanensis in China. Three rrnS sequences of S. phy. physalis (PQ818928–PQ818930) obtained herein are all 439 bp in length, with no nucleotide polymorphism detected. Pairwise comparison of the newly sequenced rrnS data of S. phy. physalis (PQ818928–PQ818930) with that of S. phy. bazhaiensis obtained herein, and S. phy. physalis (OP330326) and S. numidica (OP331197, OP331198) available in GenBank displayed 5.01%, 0.91%, and 10.5% nucleotide divergence, respectively.

ASAP and BI analyses

The ASAP and BI results based on the mitochondrial cox1, cox2, and rrnS data all supported the species partition of S. numidica, S. phy. physalis, and S. phy. bazhaiensis n. subsp. However, the present results of ASAP based on the ITS data did not support that S. phy. physalis and S. phy. bazhaiensis represent two distinct taxa (Fig. 5). The results of analyses using the cox1, cox2, and rrnS data all displayed S. numidica, S. phy. physalis, and S. phy. bazhaiensis n. subsp. as representing three distinct lineages, but the BI results based on the ITS data showed samples of S. phy. physalis and S. phy. bazhaiensis n. subsp. mixed together (Fig. 6).

thumbnail Figure 5

Assemble species by automatic partitioning (ASAP) analyses of Seuratascaris spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: cox1, cytochrome c oxidase subunit I; cox2, cytochrome c oxidase subunit II; rrnS, Small ribosomal RNA; ITS, internal transcribed spacer; OG, out-group. Asterisk indicated the optimal results recommended by ASAP.

thumbnail Figure 6

Bayesian inference of Seuratascaris spp. based on different nuclear and mitochondrial genetic markers. Bayesian posterior probability values ≥ 0.80 are shown in the phylogenetic trees.

Characterization of the complete mitogenome

The circular mitogenome of S. phy. bazhaiensis n. subsp. is 13,628 bp in length, and contains 36 genes, including 12 PCGs (missing atp8) (cox1–3, cytb, nad1–6, nad4L, and atp6), 22 tRNA genes and 2 rRNA genes (rrnL located between tRNA-His and nad3, and rrnS located between tRNA-Glu and tRNA-Ser2) (Fig. 7; Table 2). There is only 1 non-coding region (340 bp) in the mitogenome of S. phy. bazhaiensis, located between tRNA-Ser2 and tRNA-Asn. (Fig. 7; Table 2). All genes are transcribed from the same DNA strand. The overall A + T content in the mitogenome of S. phy. bazhaiensis is 73.4%, showing a strong nucleotide compositional bias toward A + T (Table 3). The length of each gene and nucleotide contents of the S. phy. bazhaiensis mitogenome are provided (Tables 2 and 3).

thumbnail Figure 7

Gene map of the mitochondrial genome of Seuratascaris physalis bazhaiensis n. subsp. All 22 tRNA genes are nominated by the one-letter coding with numbers differentiating each of the two tRNAs, serine and leucine.

Table 2

Annotations and gene organization of Seuratascaris physalis bazhaiensis n. subsp.

Table 3

Base composition and skewness of Seuratascaris physalis bazhaiensis n. subsp.

The total size of the 12 PCGs of S. phy. bazhaiensis mitogenome is 10,326 bp (excluding termination codons), ranged in length from 273 bp (nad4L) to 1584 bp (nad5), which encode 3442 amino acids, respectively (Tables 2 and 3). Among the 12 PCGs, 6 genes (cox3, nad1–2, nad4, nad6, and atp6) used TTG as the start codon, whereas 5 genes (cox1–2, nad3, nad5, and cytb) used ATT. Only nad4L used ATA as the start codon. TAG was the most commonly used termination codon for 7 genes, including cox1, cox3, nad1–3, nad4L, and nad5. Three genes (cox2, nad4, and nad6) used TAA, and the incomplete termination codon T was inferred for atp6 and cytb (Table 2). The components and usages of codons in the mitogenome of S. phy. bazhaiensis are provided in Figure 8 and in Table 2.

thumbnail Figure 8

Relative synonymous codon usage (RSCU) of Seuratascaris physalis bazhaiensis n. subsp. Codon families (in alphabetical order) indicated below the horizontal axis. Values at the top of each bar represent amino acid usage in percentage.

The gene arrangement of 36 genes in the mitogenome of S. phy. bazhaiensis belong to the GA3 type for the mitogenome of Nematoda, and is in the following order: cox1, tRNA-Cys, tRNA-Met, tRNA-Asp, tRNA-Gly, cox2, tRNA-His, rrnL, nad3, nad5, tRNA-Ala, tRNA-Pro, tRNA-Val, nad6, nad4L, tRNA-Trp, tRNAGlu, rrnS, tRNA-Ser2, tRNA-Asn, tRNA-Tyr, nad1, atp6, tRNA-Lys, tRNA-Leu2, tRNA-Ser1, nad2, tRNA-Ile, tRNA-Arg, tRNA-Gln, tRNA-Phe, cytb, tRNA-Leu1, cox3, tRNA-Thr, nad4 (Fig. 7).

Discussion

Some recent studies proved that it is powerful and practical to utilize the nuclear ITS and mitochondrial cox1, cox2, and rrnS regions as genetic markers for species identification of ascaridoid nematodes [1, 3, 9–11, 19, 23]. The present study revealed the presence of remarkable morphological variability in the lengths of the body, oesophagus, and intestinal caecum, and the number and arrangement of precloacal papillae in S. phy. physalis among different individuals collected from different or the same hosts (see Table 1 for details). However, a genetic comparison of the different samples of S. phy. physalis collected from three different frog hosts Quasipaa exilispinosa, Odorrana tiannanensis, and Rhacophorus sp., exhibited low levels of nucleotide variation in the ITS (0%), cox1 (0–2.08%), cox2 (0–2.09%), and rrnS regions (0–0.91%), which are distinctly lower than those between S. phy. physalis and S. numidica (5.55% in ITS, 12.5–12.8% in cox1, 11.1–11.2% in cox2, and 10.5% in rrnS). The present molecular evidence indicated that: (i) the newly collected nematode material from O. tiannanensis and Rhacophorus sp. in Yunnan Province belongs to S. phy. physalis; (ii) the broad ranges of morphological variability in the above-mentioned respects in different individuals of S. phy. physalis collected from different frog hosts should be considered as intraspecific variation; (iii) S. phy. physalis represents a distinct taxon from S. numidica. ASAP and BI analyses based on different nuclear and mitochondrial data supported the present results.

In the present study, we found the presence of differentiable features in the tail length and the ratio of oesophageal length to body length in the male, different geographical distribution areas (Bazhaigou in Guangxi Zhuang Autonomous Region vs. Mengla in Yunnan Province), and relatively high level of nucleotide divergence in mitochondrial sequence data (5.73–6.25% in cox1, 8.18–8.58% in cox2, and 4.51–5.01% in rrnS), between the present Seuratascaris specimens collected from O. graminea in Guangxi and S. phy. physalis in Yunan. Additionally, the present ASAP and BI results based on the cox1, cox2, and rrnS sequence data also supported the species partition of these Seuratascaris specimens and S. phy. physalis. However, we considered that the justification for describing or identifying a new species of zooparasitic nematodes should be based on enough evidence including at least the following four aspects [21], which are the data used to test species boundaries and to delimit the species: (i) presence of morphological characters with taxonomic significance for differentiating the new nominal taxon from its closest related or similar taxa; (ii) presence of ecological differences representing the potential for natural reproductive isolation (i.e., biological, geographical, and host differences); (iii) presence of enough nucleotide variation in some mitochondrial genetic makers (i.e., cox1, cox2, rrnS) representing matrilineal divergence; and (iv) presence of enough nucleotide variation in some nuclear genetic makers (i.e., ITS, 28S) representing patrilineal divergence. Consequently, it seems not clearly evident to erect a new species for our Seuratascaris specimens collected from O. graminea in Guangxi, because there is no nucleotide divergence in nuclear ITS data detected between the present material and S. phy. physalis. However, we prefer to propose a new subspecies S. phy. bazhaiensis for the present Seuratascaris material collected from O. graminea in Guangxi, which can be discriminated from the S. phy. physalis based on the differences in morphology, genetics (mtDNA), and geography. Seuratascaris physalis bazhaiensis and S. phy. physalis may represent “species on the road to differentiation”.

Liu et al. (2023) [17] reported S. schmackeri Liu, Fang, Zheng & Wu, 2023 from Odorrana schmackeri Boettger (Anura: Ranidae) in China, and also provided the ITS (MT434777) and cox1 (MN120313) sequence data for S. schmackeri. However, the description of S. schmackeri is rather poor and, in some cases, the actual generic features are not clear, and some morphometrics are almost certainly erroneous. Additionally, molecular analysis of the genetic data displayed about 98.6% similarity in the ITS region between S. schmackeri and Megalobatrachonema hainanensis (Cosmocercoidea: Kathlaniidae) (MH545567, MH545568). Furthermore, according to Liu et al. (2023) [17], the presence of over 50.0% nucleotide divergence in the cox1 sequence between S. schmackeri and S. numidica also indicated that S. schmackeri is not a member of the genus Seuratascaris. We made a request to borrow the type material of S. schmackeri deposited in Wanna Medical College, China, but failed. Consequently, in order to avoid confusion of the taxonomy of Seuratascaris, S. schmackeri should be considered as incertae sedis or species inquirenda.

The current mitogenomic database for ascaridoid nematodes remains insufficient. To date, a total of 33 ascaridoid species with their mitogenomic data are available in GenBank or other databases, but there are no mitochondrial genomic data of Seuratascaris species reported so far. The mitogenomes of ascaridoid nematodes seem to exhibit a high degree of conservation in the composition and gene arrangement order. The composition [including 12 PCGs (missing atp8), 22 tRNA genes, and 2 rRNA genes] and gene arrangement type (GA3 type) of S. phy. bazhaiensis mitogenome agreed well with those of the reported mitogenomes of ascaridoid nematodes [6, 7, 10, 13, 16, 20, 26–31], but S. phy. bazhaiensis has only one non-coding region in the mitogenome, which is different from all of the reported ascaridoid mitogenomes, except Ortleppascaris sinensis [30]. The size of the complete mitogenome of S. phy. bazhaiensis (13,628 bp) represents the smallest mitogenome in the reported ascaridoid mitogenomes so far (13,828–15,045 bp). The mitogenome of S. phy. bazhaiensis displayed a strong nucleotide compositional bias toward A + T (73.4%), which is higher than all of the reported mitogenomes of ascaridoid nematodes, except O. sinensis (74.0%) [30]. The present study enriched the mitogenomic data and revealed the pattern of mitogenomic evolution of ascaridoid nematodes.

Conclusion

Seuratascaris physalis bazhaiensis n. subsp. was described based on integrated evidence. ASAP and BI results based on the mitochondrial cox1, cox2 and rrnS data all supported S. phy. bazhaiensis representing a distinct taxon from S. phy. physalis. The supplementary morphometric and genetic data of S. phy. physalis based on newly collected specimens from O. tiannanensis and Rhacophorus sp. would enable us to diagnose this subspecies more accurately. A key to species of Seuratascaris is provided. The complete mitochondrial genome of S. phy. bazhaiensis was also sequenced and annotated, which represents the first mitogenomic data for the genus Seuratascaris. The mitogenome of S. phy. bazhaiensis has only 13,628 bp (including 12 protein-coding genes, 22 tRNA genes, 2 ribosomal RNAs, and only 1 non-coding region), and represents the smallest mitogenome of the reported ascaridoid mitogenomes so far.

Acknowledgments

The authors are grateful to Dr. Hui Wang (Hebei Normal University, China) for collecting and identifying frogs.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 32170442), and the Key Development Foundation of Hebei Normal University (L2024ZD17) for Dr. Liang Li.

Conflicts of interest

The authors declare that they have no competing interests.

Author contribution statement

X.-H. G. and L.L. contributed to the study design, annotated mitogenome, and conducted the ASAP and BI analyses. X.-H. G. J.-T. M. and H.-X. C. identified nematode specimens, sequenced and analysed genetic data. X.-H. G., J.-T. M., H.-X. C. and L.L. wrote the manuscript. All authors read and approved the final manuscript.

a

Xiao Hong Gu and Jia-Tong Mu contributed equally to this work.

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Cite this article as: Gu X-H, Mu J-T, Chen H-X & Li L. 2025. Integrated evidence reveals a new subspecies of the genus Seuratascaris (Nematoda: Ascaridomorpha), with characterization of the complete mitochondrial genome. Parasite 32, 14. https://doi.org/10.1051/parasite/2025008.

All Tables

Table 1

Morphometric comparisons of Seuratascaris physalis bazhaiensis and Seuratascaris physalis physalis. Measurements are given in micrometers (μm).

In the text
Table 2

Annotations and gene organization of Seuratascaris physalis bazhaiensis n. subsp.

In the text
Table 3

Base composition and skewness of Seuratascaris physalis bazhaiensis n. subsp.

In the text

All Figures

thumbnail Figure 1

Scanning electron micrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China, male. A: anterior part of body (excretory pore arrowed), lateral view; B: magnified image of excretory pore, ventral view; C: magnified image of excretory pore, lateral view; D: dorsal lip; E: subventral lip; F: tail (postcloacal papillae indicated using white arrows, phasmid indicated using black arrow), ventral view; G: magnified image of precloacal papillae; H: magnified image of medio-ventral precloacal papilla; I: magnified image of postcloacal double papillae; J: magnified image of postcloacal papillae; K: posterior end of body (precloacal papillae arrowed), ventral view; L: magnified image of tail tip. Abbreviations: am, amphid; dp, double papilla; sp., single papilla.
In the text
thumbnail Figure 2

Scanning electron micrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China, female. A: cephalic end, apical view; B: cephalic end, lateral view; C: magnified image of denticles; D: magnified image of tail tip (phasmids arrowed); E: posterior end of body, ventral view; F: region of vulva, ventral view.
In the text
thumbnail Figure 3

Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China. A: anterior part of male body, lateral view; B: cephalic end of male, dorsal view; C: cephalic end of male, sublateral view; D: region of vulva, lateral view; E: tail of female, lateral view; F: egg; G: posterior end of male, ventral view; H: spicules; I: posterior end of male, lateral view.
In the text
thumbnail Figure 4

Photomicrographs of Seuratascaris physalis bazhaiensis n. subsp. collected from Odorrana graminea (Boulenger) (Anura: Ranidae) in China. A: anterior part of male, lateral view; B: cephalic end of male, dorsal view; C: cephalic end of male, ventral view; D: region of vulva, lateral view; E: posterior end of female, lateral view; F: posterior end of male, lateral view; G: posterior end of male, ventral view.
In the text
thumbnail Figure 5

Assemble species by automatic partitioning (ASAP) analyses of Seuratascaris spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: cox1, cytochrome c oxidase subunit I; cox2, cytochrome c oxidase subunit II; rrnS, Small ribosomal RNA; ITS, internal transcribed spacer; OG, out-group. Asterisk indicated the optimal results recommended by ASAP.
In the text
thumbnail Figure 6

Bayesian inference of Seuratascaris spp. based on different nuclear and mitochondrial genetic markers. Bayesian posterior probability values ≥ 0.80 are shown in the phylogenetic trees.
In the text
thumbnail Figure 7

Gene map of the mitochondrial genome of Seuratascaris physalis bazhaiensis n. subsp. All 22 tRNA genes are nominated by the one-letter coding with numbers differentiating each of the two tRNAs, serine and leucine.
In the text
thumbnail Figure 8

Relative synonymous codon usage (RSCU) of Seuratascaris physalis bazhaiensis n. subsp. Codon families (in alphabetical order) indicated below the horizontal axis. Values at the top of each bar represent amino acid usage in percentage.
In the text

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