Seuratascaris schmackeri sp. nov. (Nematoda: Ascarididae) from the Chinese Frog Odorrana schmackeri Boettger, 1892 (Amphibia: Anura) Based on Morphological and Molecular Evidence

Ying Liu1,2, Ji-Yong Fang2, Na Zheng3 and Hai-Long Wu1*

1Anhui Provincial Key Laboratory of Biotic Environment and Ecological Safety, College of Ecology and Enviroment, Anhui Normal University, Wuhu, 241000, Anhui Province, People’s Republic of China.

2School of Basic Medical Sciences, Wannan Medical College, Wuhu, 241000, Anhui Province, People’s Republic of China.

3School of Clinical Medical Sciences, Wannan Medical College, Wuhu, 241000, Anhui Province, People’s Republic of China.

ABSTRACT

Here, we examined the sequences of the internal transcribed spacer1 (ITS1) region, the partial small ribosomal RNA gene (18S), and mitochondrial cytochrome-c oxidase subunit 1 (COI) genes of Seuratascaris schmackeri sp. nov., a member of ascaridoids, exhibiting characteristics of Ascaridoidea sensu Chabaud (1965) identified through microscopy. This new species was collected from the small intestine of the Chinese frog species Odorrana schmackeri Boettger, 1892, acquired from four regions of Anhui Province, southeastern China. To our knowledge, one species of the Seuratascaris genus, namely Seuratascaris numidica (Seurat, 1917) Sprent 1985, has so far been recorded. The morphology of S. schmackeri sp. nov. differs from that of S. numidica and exhibit a few unique characteristics, including more denticles in the lip, shorter intestinal caecum, longer spicular, more caudal papillae, and pre-, ad-, and post-cloacal caudal papillae pairs in the ratio of 3: 1: 6-7. BLAST analyses of the COI sequences show 59.31% nucleotide divergence with Seuratascaris numidica (Seurat, 1917) (GenBank acc. no. MG434691 and MG434692). Through morphological and molecular characterization of S. schmackeri sp. nov., we generated new data on the Seuratascaris genus, providing a crucial scientific basis for future studies on the genus.

Article Information

Received 25 May 2022

Revised 03 June 2022

Accepted 14 June 2022

Available online 20 September 2022

(early access)

Published 05 June 2023

Authors’ Contribution

Ying Liu, Species identification;

Ji-Yong Fang, Sequence Alignment;

Na Zheng, DNA extraction;

Hai-Long Wu, Professional guidance.

Key words

Ascarididae, Odorrana schmackeri, Morphological evidence, Molecular evidence, Seuratascaris schmackeri sp. nov.

DOI: https://dx.doi.org/10.17582/journal.pjz/20220525160559

* Corresponding author: [email protected]

0030-9923/2023/0004-1621 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

A new genus, Seuratascaris Sprent, 1985 initially described as Porrocaecum numidicum, was discovered from a frog species, Pelophylax saharicus (syn. Rana saharicus), collected in Algeria by Seurat (1917). Remarkable morphological variability was noted among the specimen isolated from various frogs and toads in different geographical locations worldwide, including Mediterranean specimens and South East Asia specimens (Sprent, 1985). Sprent also considered that the species identified by Le Van Hoa (1960), namely numidicum Seurat, 1917, ranae Gupta, 1959, cacopi Chatterji, 1936, and Amplicaecum communis Yuen, 1963, belong to the genus of Seuratascaris and are merged to a single species. The type species, S. numidica. Seuratascaris, a parasite of amphibians, is uniquely distinguished from other ascarids by intestinal caecum, presence or absence of interlabial, the absence of an oesophageal appendix, and lateral alae, the sort and number of caudle papillae, and a distinctive blunt end of an immature Seuratascaris.

Recently, Chen et al. (2018) provided the morphological characterization and molecular evidence of S. numidica from the frog, Hoplobatrachus chinensis (Osbeck, 1765), in China. However, due to a lack of genetic data, Chen and colleagues could not confirm whether S. numidica is a single species or not. Wang et al. (1981) had reported Angusticaecum wuyiensis, characterized by longer intestinal caecum, precolocal (5-6 pairs), postcolocal (4 pairs), large preanal central papilla, from Rana schmackeri Boettger,1892 (Syn. Odorrana schmackeri Boettger, 1892) in Wuyi Mountain, Fujian Province, China. According to Chen et al. (2018), A. wuyiensis Wang, 1981 is a novel synonym of Seuratascaris numidica.

The present study morphologically recognized several Ascarididae nematodes collected from the intestine of Odorrana schmackeri in Anhui Province, China, as species in the genus Seuratascaris. Detailed evidence of molecular characterization of the ITS, COI, and 18S regions of the present specimen suggests a new species, designated as Seuratascaris schmackeri sp. nov.

MATERIALS AND METHODS

Collection of nematode samples and morphological observation

Eighty-eight frogs, Odorrana schmackeri, collected between June and July 2018 in the south of Anhui province, China, were examined for nematodes. Frogs were sampled from the following locations: Zhanghe (30°6’N, 118°47’E, n=22) in Jixi County, Yiwanling (30°23’N, 118°43’E, n=21), in Jingde County, Taoling (30°31’N, 118°38’E, n=13) in Jing County, Fuxi (30°4’N, 118°9’E, n=32) in Huangshan city.

Nematodes isolated from the intestine of frogs were selected and immediately immersed in 60 °C water to keep their body stretched. The worms were then fixed in 70% alcohol and disposed of gradually, awaiting subsequent experiments. The fixed nematodes were transparentized with lactic acid, phenol, and glycerin and placed under a light microscope (Olympus BX51FL-DIC) to examine the internal anatomy structure.

The morphological characteristics of the nematode body surface were assessed by a scanning electron microscope (SEM). Briefly, two females and two male specimens were pre-fixed in 3.0% glutaraldehyde, later fixed in 1% OsO4, and processed via gradient alcohol dehydration. The specimens were gold-coated and examined by Gemini SEM 300 (Carl Zeiss, Germany) at the accelerating voltage of 20KV. The diagram was made with the aid of a Nikon mi-manually depending on the light microscopy images. Measurements were recorded in millimeters (mm) unless diversely verbalized. Voucher specimens were stored in the Medical Parasitology Department of Wannan Medical College in Anhui Province of China.

DNA extraction and sequencing analyses

Three specimens were selected randomly for sequencing of ITS, COI, and 18S regions. Briefly, genomic DNA of the helminth was extracted using a TIANamp Genomic DNA Kit (TianGen Biotech, Beijing, China) following the manufacturer’s instructions. The target regions were amplified by polymerase chain reaction (PCR) using the following primers pairs: ITS1 (Gasser et al., 1999); COI (Folmer et al., 1994); 18S (Floyd et al., 2005). The cycling conditions were as described by Li et al. (2016). The amplified products were electrophoresed on GoldView-stained 1.5% agarose gels and purified with the Column PCR Product Purification Kit (Sangon Biotech, Shanghai, China). The amplicons were sent to Sangon Biotech (Shanghai) Co. Ltd. for clone sequencing (ABI 3730, USA). Sequencing for every swatch was fulfilled for both lines. The sequences were strung out by the DNAMAN software (Lynnon Corporation, Canada) and altered in manual-acting, excluding primers, respectively. Next, the resulting sequences were compared (using the algorithm) to the available sequences in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov) using the BLAST program. All sequences of the new nematode species have been deposited in the GeneBank database (http://www.ncbi.nlm.nih.gov).

RESULTS

(Figs. 1-3)

Seuratascaris schmackeri sp. nov.

General

These are medium-length thin worms. The bodies of nematodes treated with alcohol appear white, non-transparent, and cylindrical. One-third of the middle body is the widest part. The females are larger than males. The cuticle is striped breadthwise and interspersed with slight elevation on occasion. The cephaliced features comprise three conspicuous quadrate lips with dentigerous ridges, one in dorsal position and two in latero-ventral position, broader rather than long. Whereas the dorsal lip possesses two larger external papillae, each of two latter lips have one larger papilla in ventro-lateral position and a smaller single latero-ventral papilla together with the amphidial pore in the lateral position. Interlabial is absent. Highly developed shorter postlabial grooves are found on the bottom of two neighboring lips, tied together by cuticular interlacement. The inside surface of the edged lips is jagged; the dentations are finespun, slim but protuberant, with approximately 60-70 and 90-100 denticles on each lip in female and male, respectively. Isthmus is wide (attached to the body), ventriculus, and lacks the ventricular appendix. The intestinal caecum is shorter. The intestine membrane is cast into characteristic transverse folds that give the internal surface an irised curl aspect. These spines give a herring-bone appearance of the external body layer in the side of view. The rectal gland is present. The tail is round and blunt with a small tip in two sexes. Phasmids are absent.

Male (n=3. Units of measurement in mm)

Length 5.81-12.30, maximum breadth 0.23- 0.34, head diameter 0.05- 0.08. Dorsal lip length 0.049-0.061, maximum width 0.061-0.800, interlabia absent, inconspicuous postlabial grooves (Figs. 2A, 3A), 0.014 (22.95% of max. width) in a 5.81 specimen. Pulp is not deeply divided anteriorly. Oesophagal length 1.25, shorter intestinal caecum (Fig. 1A), left 0.116 (9.3% of the length of the oesophagus), right 0.119 (9.5% of the length of the oesophagus), respectively. Pharynx and never ring from the anterior extremity of the body 0.10 and 0.45, respectively. Caudal alae are well-developed, which project 10-11 pairs of pedunculated papillae, of which 3 pairs are precloacal, 1 pair ad-cloacal, and 6-7 pairs post-cloacal, big median papillae in front of cloacal (Fig. 2B, E, F). The two spicules are equal (Fig. 2C), relatively strong, and sharpened near the tip. One of the spicules has a C-shaped wing membrane and half-wraps the other (Fig. 2C), 0.532-0.719 long. Gubernaculum is absent. The tail is blunt, 0.364-0.610 long, with a small tip.

 

 

Female (n=4, Units of measurements in mm)

Length 25.33-30.90, maximum breadth 0.70-0.74, head diameter 0.11-0.19. Latero-ventral lip length 0.093, maximum width 0.12, postlabial grooves 0.031 long (26.25% of maximum width) in a 26.84 species. Length of the oesophagus and intestinal caecum 5.23-6.89 and 0.182-0.199 (2.6-3.5% of the length of the oesophagus), respectively. Never ring from the anterior extremity of the body 1.36-1.94. Vulva at anterior half of the body, 7.74-8.94 from the anterior end and offered with two prominent lips one anterior and the other posterior. The vagina is long and muscular, running obliquely posteriorly in the body across the dorsal side of the body cavity. Uteri are long and parallel, running posteriorly. The origin of ovarian coils is a little anterior to the level of the anus. The tail is blunt, 0.127-0.224 long, with a pointed tip. The anus, 0.192-0.203 from the posterior end (Fig. 3C-D). The vagina is muscular and runs towards the posterior extremity, joining the common uterus formed by two uteri.

 

Egg (n=10, Units of measurement in mm)

Oval, eggshell with three layers: The inner membrane is extremely thin, the middle layer is thicker, concave egg covers at both ends or one end of the egg, and the outermost layer comprises proteins with uneven surface and fine particles, 0.134-0.143×0.083-0.086.

Taxonomic summary

Geographical location - Jing County, Fuxi (30°4’ N, 118°9’ E), Huangshan city, Zhanghe (30°6’ N, 118°47’ E), Jixi County, Yiwanling (30°23’ N, 118°43’ E), Jingde County, Taoling (30°31’ N, 118°38’ E), Anhui Province, China.

Host: Odorrana schmackeri Boettger, 1892, Class Amphibia, Order Anura.

Site of infection: Small intestine.

Level of infection: Total rate 11.4 % (10/88), the average and scope of infection intensity 0.58 (0.00, 11.00).

Specimens: Holotype, male (SS2018001M); allotype, female (SS2018001F); paratypes, males (SS2018002M) and females (SS2018002F). Specimens are deposited in the Medical Parasitology Department of Wannan Medical College, Wuhu, Anhui province, China.

Etymology: Based on the name of the host.

Remarks

Morphological evidence

The genus Seuratascaris, ranging from small to medium-sized ascaridoids with characteristics of Ascaridoidea sensu Chabaud (1965), was described to possess several other unique characteristics. Lips are slightly narrower than the body, with a wide isthmus (attachment to body) and dentigerous ridge all-round margin. Anterior margin has a cleft in pulp not deeply divided anteriorly. The median lobe is absent. The Interlabial region forms an interlabial ridge of variable form, cervical alae, ventriculus, and lacks a gubernaculum. Excretory pore and cervical papillae are slightly behind the nerve ring. The excretory nucleus is relatively large, situated on the left side in the commissural part of the excretory cell. The excretory system is bilateral. Intestinal caecum and rectal glands are present, male with relatively short, stout spicules, ventral cuticular precloacal ornamentation not present, female with vulva anterior to middle of body, uterus didelphic, opisthodelphic, parasites of old world anurans (Sprent, 1985).

S. schmackeri sp. nov. possesses characteristics of the genus Seuratascaris, including three dentigerous ridges around lips, wide isthmus, intestinal caecum present, gubernaculum absent, with large median papillae in front of the cloaca, and stout spicules. Seuratascaris was considered to comprise type species, S. numidica, manifesting polymorphism regarding the form of interlabial region and the number of precloacal papillae (Sprent, 1985). S. schmackeri sp. nov. poses unique morphological characteristics different from S. numidica, including shorter intestinal caecum (2.6-9.5% of oesophagus length vs 43-58% and 68.4–71.1%) (Sprent, 1985; Chen et al., 2018), longer stout spicules (0.532-0.719 vs 0.14-0.34 and 0.15-0.17) (Sprent, 1985; Chen et al., 2018) and different pairs of caudle papillae (pre-: ad-: post cloacal papillae = 3: 1: 6-7 vs 2-3, 3-5, 5-6: 0: 3-4 and 4: 0: 4-5) (Sprent, 1985; Chen et al., 2018).

Seuratascaris numidica (Seurat, 1917), from Hoplobatrachus chinensis (Osbeck) (Anura: Dicroglossidae), is the first record of the Seuratascaris genus in China (Chen et al., 2018). Seuratascaris schmackeri sp. nov. is the second record of the genus Seuratascaris in China; notably, the first record of this genus infected a Ranidae frog, O. schmackeri.

Molecular evidence

Sequences recovered from three different S. schmackeri sp. nov. specimens were identical (100% homology) for 18S rDNA, ITS and COI gene amplified fragments. Excluding primers, the18S rDNA, ITS, and COI genes of S. schmackeri sp. nov. were 885, 763, and 655 base pairs long and deposited in the GenBank database (accession nos. MN120312, MT434777, and MN120313, respectively).

No species of Seuratascaris and Angusticaecinae with 18S sequences are available in GenBank. A comparison of the 18S sequences of S. schmackeri sp. nov. with the species of Ascarididae, Baylisascaris ailuri (JN256991), B. schroederi (JN256992), B. transfuga (JN256988), Porrocaecum angusticolle (EU004820), P. reticulatum (MF072700), Parascaris equorum (U94378), Ascaris suum (MN558962), and A. sp. (JN256985) in GenBank show 5.98–6.31% nucleotide divergence.

One other species of Seuratascaris and no Angusticaecinae with ITS sequences is available in GenBank (Seuratascaris numidica, MG434689, MG434690). Pairwise comparison between S. schmackeri sp. nov. and S. numidica show 3.82% nucleotide divergence.

One other species of Seuratascaris and no Angusticaecinae with COI sequences is available in GenBank (Seuratascaris numidica, MG434691, MG434692). Pairwise comparison between S. schmackeri sp. nov. and S. numidica show 59.31% nucleotide divergence. A comparison of the COI sequences of S. schmackeri with the species of Ascarididae, Ascaris lumbricoides (AP017677, KY368757, KY368759, 045803), A. sp. (MH059555, KC839986), A. suum (KY045800), A. ovis (KU522453), Parascaris equorum (MF678786), and P. univalens (KM216010, KM067271) in GenBank show 14.16–15.29% nucleotide divergence.

DISCUSSION

The genus Seuratascaris Sprent, 1985 is discriminated from other genera of Ascaridoidea, including Amplicaecum Seurat, 1917, Orneoascaris Skrjabin, 1916, Angusticaecum Baylis, 1920, Ophidascaris Baylis, 1921 and Freitasascaris Sprent, 1983 by posterior borders of lips with wider isthmus (attachment to body) and an interlaboratory area characterized by a variable form of an interlaboratory ridge. Angusticaecum numidicum Seurat, 1917 found in anuran amphibians was re-classified to Amplicaecum Baylis, 1920 by Chabaud and Campana-Rouget (1955), subsequently to Orneoascaris Skrjabin, 1916 by Le van Hoa (1960) and finally to Seuratascaris Sprent, 1985 by Sprent (1985). S. numidica is a specialized parasitic nematode species of amphibians only (Sprent, 1985).

Species collected from frogs and toads in France and South East Asia (Mediterranean, Bali, North Borneo, Malaya, Burma, West Irian New Guinea, and Queensland) by Sprent (1985) were merged to a single species, Seuratascaris numidica (Seurat, 1917).

Seuratascaris spp. has multifarious anuran hosts. Unlike Orneoascaris spp., Seuratascaris spp. cannot extend their scope to reptiles. Literature evidence shows that S. numidica was recovered from the digestive tract of frogs in South East Asia, including stomach of the brackish water frog, Rana cancrivora Gravenhorst, 1829 from the Philippines (Bursey et al., 2003), Sylvirana supragrisea (Anura: Ranidae) from Papua New Guinea (Bursey et al., 2008), the Mao-Son frog, Hylarana maosonensis Bourret, 1937 from Vietnam (Bursey and Goldberg, 2011), Hylarana waliesa, from Papua New Guinea (Goldberg et al., 2013), intestine and stomach of the frog, Hoplobatrachus tigerinus, from India (Sou and Bursey, 2017), stomach and small intestine of Ranid frogs, Chalcorana labialis, Hylarana erythraea, and Pulchrana banjarana, all from Southeast Asia (Goldberg et al., 2017a), stomach of the toad, Phrynoidis asper, from Peninsular Malaysia (Goldberg et al., 2017b), gastrointestine of the frog, Hoplobatrachus chinensis, from Chnia (Chen et al., 2018), intestine of the frog, Limnonectes macrocephalus, from Philippines (Goldberg et al., 2019).

Seuratascaris schmackeri sp. nov. is discriminated from S. numidica by a shorter intestinal caecum and two equal longer spicules. While the Asian specimens reported by Sprent (1985) possessed paracloacal double papillae, the newly described species has one simple paracloacal papillae. Sprent (1985) and Chen et al. (2018) found that the intestine caecum length of S. numidica is not more than 60% and 68.4%-71.7% of oesophageal length, respectively, whereas that of S. schmackeri sp. nov.is only 2.6%-9.5%. The denticles on each lip of S. schmackeri sp. nov. are fine and slim but prominent, with approximately 60-70 and 90-100 denticles in females and males, respectively, differing from 64-76 denticles on each lip of S. numidica (Chen et al., 2018).

Molecular analysis of the specimens of S. schmackeri sp. nov. shows no nucleotide variation in the18S, ITS, and COI sequences. A comparison of ITS and COI sequences of S. schmackeri sp. nov. to those of S. numidica Seurat, 1917 in GenBank demonstrate 3.82% and 59.31% nucleotide divergence, respectively.

CONCLUSIONS

Morphological and genetic identification of Seuratascaris schmackeri sp. nov. from the small intestine of O. schmackeri in China is reported. The difference of morphological characteristics and the divergence of 18S, ITS, and COI between S. schmackeri sp. nov. and S. numidica Seurat, 1917 suggest that the presently described specimen is a new species of the genus Seuratascaris. S. schmackeri sp. nov. is the second species of this genus recovered worldwide.

ACKNOWLEDGEMENTS

We give sincere gratitude to Professor Chaopin Li of Wannan Medical College for his expertise in nematode identification. The authors appreciate the Laboratory of Electron Microscopy staff and the Testing Centre of Yangzhou University for their SEM technical support. We are grateful to Lisong He and Jun He for their assistance in collecting and identifying amphibian hosts. Special thanks to Huijuan Zhang, Shuaitao Deng, and Ting Liu, who dissected the frogs and classified the nematodes.

Financial support

This study was supported by the National Natural Science Foundation of China (nos: 31370537, nos: 31971199), National level College Student Innovation and Entrepreneurship Training Program (nos: 202010368058), and the Key Program Research Fund of Wannan Medical College (nos: WK2020Z06).

Statement of conflict of interest

The authors have declared no conflict of interest.

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