Introduction
The Meteterakis genus was proposed by Karve (Reference Karve1930) for nematodes Meteterakis govindi Karve, Reference Karve1930 described from the intestine of Duttaphrynus melanostictus (Schneider, 1799) (as Bufo melanostictus Schneider, 1799) (Anura: Bufonidae) and placed in Heteterakinae (Nematoda: Heterakidae). Later, the genus was moved to Heterakis (Baylis, Reference Baylis1936; Koo, Reference Koo1939), and then transferred into subfamily Spinicaudinae (López-Neyra, Reference López-Neyra1947) or considered as a junior synonym for Ganguleterakis Lane, 1914 (Skrjabin & Schikhobalova, Reference Skrjabin and Schikhobalova1949). Freitas (Reference Freitas1956) reinstated Meteterakis and included it in the family Heterakidae, whereas Inglis (Reference Inglis1957a) established the subfamily Meteterakinae.
Inglis (Reference Inglis1958) made a significant revision of the Meteterakis genus having included in it eight species that previously considered as members of the following genera: Spinicauda Travassos, 1920 (i.e., M. cophotis [Baylis, 1935] Inglis, Reference Inglis1958, M. japonica [Wilkie, Reference Wilkie1930] Inglis, Reference Inglis1958 and M. longispiculata [Baylis, Reference Baylis1929] Inglis, Reference Inglis1958); Africana Travassos, 1920 (M. mabuyi [Chakravarty, Reference Chakravarty1944]) Africana varani (Chakravarty, Reference Chakravarty1944) and A. howardi Li, Reference Li1933 were considered as synonyms of M. govindi and M. japonica, respectively) and Ganguleterakis (M. triaculeata [Kreis, 1933] Inglis, Reference Inglis1958). This taxonomic decision significantly expanded the morphological scope of the genus by adding species with spicules of different sizes and morphology, as well as the number of genital papillae in the males. Inglis (Reference Inglis1958) considers one of the key characteristics of the genus to be the presence of indefinite gubernacular mass developed from the cloacal walls and present in all species (except M. triaculeata). Baker (Reference Baker1984) analyzed the composition and geographic distribution of the studied genus and noted 16 species and their synonyms. In his work, Baker (Reference Baker1984) does not attempt to resolve taxonomic issues of the genus and points out additional problems related to the outdated classification of host species. In the latest study of the genus by Junker et al. (Reference Junker, Mariaux, Measey and Mutafchiev2015), the authors mention 23 species and divide Meteterakis species into four morphological groups based on the size and the ratio of spicule lengths. The present study provides new morphological and molecular data for Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972, originally described from the intestine of Mauremys mutica (Cantor, 1842) (as Clemmys mutica Boettger, 1888) (Testudines: Geoemydidae) in Vietnam; composition of the genus Meteterakis is discussed following the analysis of the literature and the newly received data.
Materials and methods
Morphological observations
Nematodes were recovered from intestine of a female specimen of Geoemyda spengleri (Gmelin, 1789) (Geoemydae), which was kept in the Moscow Zoo Exotarium and originated from Guangxi Province (China). The exact locality is unknown. A post-mortem examination and dissection were done by Professor A.V. Tchesunov from Moscow State University. The nematode material obtained was passed to the Laboratory of Systematics and Evolution of Parasites of A.N. Severtsov Institute of Ecology and Evolution RAS for further study. Fourteen specimens were fixed in 4% formaldehyde, and 10 of them were processed to anhydrous glycerin (Seinhorst, Reference Seinhorst1959) for permanent mounts on glass slides and further observation at Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan). Two male and two female specimens were processed for scanning electron microscopy (SEM) by serial dehydrations utilizing ethanol and acetone and then dried in a Hitachi Critical Point Dryer HCP-1 (Hitachi Ltd., Japan). Nematodes were placed on aluminum stubs, and sputter-coated with gold in a S150A Sputter Coater (Edwards, UK). Specimens were viewed with a JSM-6380LA (JEOL) SEM (JEOL, Peabody, USA).
Molecular profiles
Two males and the single female specimen were fixed in 95% ethanol and stored at –18°C before DNA extraction. The DNA was extracted with the aid of QiAamp Miсro Kit (Qiagen, Germantown, USA) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the Encyclo Plus PCR kit (Evrogen, Moscow, Russia) according to the manufacturer’s instructions. Primer pairs LSU391F (5′-AGC GGA GGA AAA GAA ACT AA-3′) and LSU501R (5′-TCG GAA GGA ACC AGC TAC TA-3′) were used to amplify the D2-D3 expansion segment of the 28S rDNA fragment (Nadler et al., Reference Nadler, De Ley, Mundo-Ocampo, Smythe, Stock, Bumbarger, Adams, Tandingan De Ley, Holovachov and Baldwin2006). PCR cycling parameters included primary denaturation at 94°C for 3 minutes followed by 34 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 1 minute, followed by post-amplification extension at 72°C for 7 minutes. A pair of nematode-specific primers designated as nem18SF (5′-CGC GAA TRG CTC ATT ACA ACA GC-3′) and nem18SR (5′-GGG CGG TAT CTG ATC GCC-3′) were used to amplify the 5′ portion of 18S rDNA (Floyd et al., Reference Floyd, Rogers, Lambshead and Smith2005). PCR cycling parameters included primary denaturation at 95°C for 5 minutes followed by 5 cycles at 94°C for 30 s, 47°C for 30 s and 72°C for 40 s and 35 cycles at 94°C for 25 s, 54°C for 30 s and 72°C for 40 s, followed by post-amplification extension at 72°C for 5 minutes. Another primer pair designated as 24F (5′-AGR GGT GAA ATY CGT GGA CC-3′) and Q39 (5′-TAA TGA TCC WTC YGC AGG TTC ACC TAC-3′) were used to obtain the remaining 3′ end of 18S rDNA (Blaxter et al., Reference Blaxter, De Ley, Garey, Liu, Scheldemann, Vierstraete, Vanfleteren, Mackey, Dorris and Frisse1998). PCR cycling parameters included primary denaturation at 95°C for 5 minutes followed by 35 cycles at 94°C for 60 s, 53°C for 90 s and 72°C for 90 s, followed by post-amplification extension at 72°C for 6 minutes.
PCR reaction products were visualized in an agarose gel and bands were excised for DNA extraction and cleaned with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Ethanol precipitation was used to purify and concentrate obtained DNA. Samples were directly sequenced using the same primers as used for primary PCR reactions. Nematode sequences obtained during this study have been deposited in GenBank (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov/) as PP571902-PP571904 for 18S and PP571899-PP571901 for 28S rDNA.
For comparative purposes and phylogeny construction 28S rDNA sequences from the National Center for Biotechnology Information GenBank database were also used (GenBank accession numbers are given in the phylogram). Sequence alignments were generated using Clustal_X (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997) under default values for gap opening and gap extension penalties. MEGA 5.2 (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011) was used to select the best evolution model (J2+G) and to infer the phylogeny based on maximum likelihood method. Support for the clades of the maximum likelihood tree was assessed using bootstrap analysis with 1,000 pseudo-replicates.
Results
Family Heterakidae Railliet & Henry, 1912
Subfamily Meteterakinae Inglis, 1957
Meteterakis Karve, Reference Karve1930
Syn. Cometeterakis Cruz & Ching, Reference Crusz and Ching1975
Heterakoides Teixeira de Freitas, Reference Freitas1956
Pareterakis Teixeira de Freitas, Reference Freitas1956
Diagnosis (sensu Inglis, Reference Inglis1958, amended): Meteterakinae. Medium-sized nematodes with well-defined cuticular annulation of the body. Multiple somatic papillae dispersed throughout the body. Head end is rounded, slightly dimorphic in males and females; oral opening tri-radiate surrounded with three lips, each having a prominent cuticular flange projecting anteriorly above the lip. Three anteriorly directed pharyngeal teeth present. Six tiny papillae of inner circle and four large submedian papillae present, located as two on dorsal and one on each subventral lips. Amphid openings are slit-like and locate at oval amphidial plates. Pharynx divided into short anterior procorpus separated from the long posterior corpus by valves, isthmus and a pear-like basal bulb. The intestine is wider in its anterior part then gradually narrows posteriorly where joins the rectum. Lateral alae present; in males, it terminates at the level of the precloacal sucker, and in females continues to the end of the tail tip. The nerve ring encircles the oesophageal corpus anterior to excretory pore which opens ventrally and leads to a large excretory vesicle. Males possess precloacal ventral sucker surrounded by a well cuticularized rim and caudal alae supported by three or four large fleshy papillae. Of these papillae, two or three pairs lie about the level of the precloacal sucker and the remaining pair is lateral to the cloacal opening. Genital papillae significantly vary in number (from 6 to 23 pairs). Spicules paired, equal or subequal, alate or non-alate, usually well tessellated. Gubernaculum present. Unpaired accessory cuticular structure (the ‘scutum’), present at the cloacal aperture (see Discussion). Tail conical with narrow distal tip. In females, the vulva is located at the middle of the body and opens on its ventral side; vulval flap developed from anterior vulval lip may present or absent. Genital tract didelphic, opistodelphic. Vulva leads to a short vagina vera which turns posteriorly and runs into a long vagina uterina, which in turn continues posteriorly as a common uterus and splits into two parallel uteri. Tail long, conical, sharply pointed. Mature eggs are oval with thick and smooth shells.
Type species:
Meteterakis govindi Karve, Reference Karve1930
syn. Africana varani Maplestone, 1931
Spinicauda bufonis Yamaguti, Reference Yamaguti1935
Heterakis govindi Baylis, Reference Baylis1936
Ganguleterakis govindi Skrjabin, Reference Skrjabin and Schikhobalova1949
Meteterakis gambhiri Zhang & Zhang, Reference Zhang and Zhang2011 (see Comments section in current paper)
Meteterakis bufonis Gambhir et al., Reference Gambhir, Tarnita, Chinglenkhomba, Gyaneswori and Indrani2006 (see Comments section in current paper)
Other species:
Meteterakis amamiensis Hasegawa, Reference Hasegawa1990
Meteterakis asansolensis Sou, Reference Sou2019
Meteterakis andamanensis Soota & Chaturvedi, 1972
Meteterakis aurangabadensis Deshmukh & Choudhari, 1980
Meteterakis baylisi Inglis, Reference Inglis1958
Meteterakis formosensis Sata, Reference Sata2018
Meteterakis guptai Gupta & Naiyer, Reference Gupta and Naiyer1993
Meteterakis hurawensis Bursey et al., 2017
Meteterakis ishikawanae Hasegawa, Reference Hasegawa1987
Meteterakis japonica (Wilkie, Reference Wilkie1930) Inglis, Reference Inglis1958
syn. Spinicauda japonica Wilkie, Reference Wilkie1930
Africana howardi Li, Reference Li1933
Pareterakis howardi (Li, Reference Li1933) Freitas, Reference Freitas1956
Meteterakis howardi (Li, Reference Li1933) Skrjabin, Schikhobalova & Lagodovskaja, Reference Skrjabin, Schikhobalova and Lagodovskaya1961 (see Comments section in current paper)
Meteterakis karvei Naidu & Thakare, 1981
Meteterakis lombokensis Purwaningsih, Dewi & Nugroho, Reference Purwaningsih, Dewi and Nugroho2016
Meteterakis longispiculata (Baylis, Reference Baylis1929)
syn. Spinicauda longispiculata Baylis, Reference Baylis1929
Meteterakis louisi Inglis, Reference Inglis1958
Meteterakis occidentalis Sata, Reference Sata2018
Meteterakis paucipapillosa Wang, 1980
Meteterakis pursatensis Bursey, Goldberg & Grismer, 2019
Meteterakis saotomensis Junker, Mariaux, Measey & Mutafchiev, Reference Junker, Mariaux, Measey and Mutafchiev2015
Meteterakis singaporensis (Sandosham, 1954)
Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972
Meteterakis vaucheri Adamson, Reference Adamson1986
Meteterakis wangi Zhang & Zhang, Reference Zhang and Zhang2011
Meteterakis wonosoboensis Purwaningsih, Dewi & Hasegawa, Reference Purwaningsih, Dewi and Hasegawa2015
Species inquirendae:
Meteterakis crombiei Bursey Goldberg & Kraus, Reference Bursey, Goldberg and and Kraus2005
Meteterakis lyriocephali (Crusz & Ching, Reference Crusz and Ching1975)
Meteterakis mabuyi (Chakravarty, Reference Chakravarty1944)
syn. Africana mabuyae Chakravarty, Reference Chakravarty1944
Meteterakis sinharajensis (Crusz & Ching, Reference Crusz and Ching1975)
Meteterakis triaculeata (Kreis, 1933)
syn. Ganguleterakis triaculeatus Kreis, 1933
Species incertae sedis:
Africana bufonis Biswas & Chakravarty, 1963
Meteterakis bufonis (Biswas & Charkavarty, 1963) Baker, Reference Baker1984
syn. Heterakis bufonis Biswas & Charkavarty, 1963
Comments:
Meteterakis gambhiri Zhang & Zhang, Reference Zhang and Zhang2011 is a replacement name for Meteterakis bufonis Gambhir et al., Reference Gambhir, Tarnita, Chinglenkhomba, Gyaneswori and Indrani2006, which, according to the International Code of Zoological Nomenclature (ICZN), was a junior secondary homonym of Meteterakis bufonis (Biswas & Charkavarty, 1963) Baker, Reference Baker1984 (see Baker; Reference Baker1984; ICZN, 1999 Articles 57.3 and 72.7; Zhang & Zhang, Reference Zhang and Zhang2011).
Meteterakis howardi (Li, Reference Li1933) was accepted by Skrjabin et al. (Reference Skrjabin, Schikhobalova and Lagodovskaya1961) as an independent representative of Meteterakis described as Africana howardi by Li (Reference Li1933), while Inglis (Reference Inglis1958) recognized this species as a junior synonym for Meteterakis japonica (Wilkie, Reference Wilkie1930). Inglis’s opinion has become established in the literature, and the name combination Meteterakis howardi has never been mentioned in further revisions of the genus (Baker, Reference Baker1984; Junker et al., Reference Junker, Mariaux, Measey and Mutafchiev2015).
Meteterakis (Spinicauda) mathevossianae (Skarbilovich, Reference Skarbilovich1950) was mentioned in the description of M. bufonis (Gambhir et al., Reference Gambhir, Tarnita, Chinglenkhomba, Gyaneswori and Indrani2006), however, it is still considered a valid species of the genus Spinicauda (Nematoda: Heterakidae) (see Jamdar, Reference Jamdar2021; Gambhir et al., Reference Gambhir, Tarnita, Chinglenkhomba, Gyaneswori and Indrani2006; Skarbilovich, Reference Skarbilovich1950).
Meteterakis rodriguesi Vicente & Corrêa Gomes, 1971 has been accepted as Bufonerakis rodriguesi (Vicente & Corrêa Gomes, 1971) (see Baker, Reference Baker1980).
Hosts and habitat:
Gastrointestinal tract of reptiles and amphibians.
Geographical distribution:
India, Sri Lanca, Myanmar, Vietnam, China, Japan, East Indies, and Afrotropics.
Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972 (Figures 1–7)
Line drawings of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972. (A) male, entire view; (B) female, entire view; (C) female anterior end, lateral view; (D) vulva and anterior part of female genital tract, ventral view; (E, F) vulva, lateral view (right and left sides, respectively); (G) female posterior region, lateral view. (Scale bars for A = 35 μm, B = 600 μm, C = 150 μm, D = 135 μm, E = 100 μm, F = 125 μm, G=85 μm.)
Measurements
See Table 1.
Morphometrics (in μm unless otherwise stated) of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972 (present study and the original description) and Meteterakis ishikawanae Hasegawa, Reference Hasegawa1987; mean ± standard deviation (range), except for de Man ratios
a total body length divided by maximum body diameter;
b total body length divided by pharyngeal length (the pharynx is defined as head end to the pharyngo-intestinal junction);
c total body length divided by tail length;
c′ tail length divided by body diameter at the anal/ cloacal aperture;
L total body length (head to tail tip);
L′ body length from head to anal/ cloacal aperture; n, number of specimens examined;
V position of vulva from anterior end expressed as percentage of body length;
V′ position of vulva from anterior end expressed as percentage of distance from head to anal aperture; data for holotype specimen of M. striatura is given in bold.
* Six nematode specimens have been studied from the same host; however, the exact number of measured male specimens is not in the main description. The holotype female and one paratype female specimens containing mature eggs have been measured.
** Measurements holotype male and allotype female are not given separately.
General
Body cuticle with fine striation beginning just posterior the cephalic papillae and continuing nearly to the end of tail (Figure 3D). Tiny somatic papillae are scattered throughout the body. Prominent lateral alae present. Distal edges of the alae are bifid in transverse section. Cephalic extremity rounded with tri-radiate oral opening, surrounded with three well-developed lips each with prominent cuticular flange projecting anteriorly (Figure 3A; Figure 5A, B). Six tiny papillae of inner circle present as two on each lip closely to oral opening. Dorsal lip is wider and bears two large double cephalic papillae, each subventral lip with one large double cephalic papilla present; slit-like amphid opening located at oval amphidial plates, and three small papillae located posterior to each amphid (Figure 3C, E, F). Anterior end of pharynx is divided into three lobes, one dorsal and two subventral; each bears anteriorly directed pharyngeal tooth, where the dorsal is the largest. Pharynx is long, and muscular, separated from the cylindrical part of the oesophagus by valves, isthmus, and a basal bulb (Figure 1A–C; Figure 2A). Isthmus is narrow, well defined from the corpus. Basal bulb pear-like, consisting of three well-defined lobes with strongly developed valves; cardia are moderately developed. The intestine is narrow at its junction with the basal bulb, expands rapidly forming a short wide part then gradually narrowing to adjoin the rectum. The nerve ring is in the mid-level of pharyngeal corpus (Figure 1A, B). Excretory pore opens on the ventral body side posterior to nerve ring and leads into the large vesicle (Figure 2B; Figure 5E, F).
Line drawings of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972, male. (A) anterior end, lateral view; (B) excretory pore, ventral view; (C) cloaca region with protruding scutum, lateral view; (D) posterior end, ventral view; (E) optical section trough cloaca region, spicules and gubernaculum disposition can be observed, lateral view; (F) posterior end, lateral view; (G) spicules distal ends and gubernaculum, ventral view. (Scale bars for A = 750 μm, B = 60 μm, C = 35 μm, D = 120 μm, E, F = 100 μm, G = 65 μm.)
SEM images of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972 (A) female anterior end, ventral view; (B) female posterior end, ventral view; (C) female head end, enface view; (D) cuticular annulation and somatic papilla, female mid-body region; (E) male head end, enface view; (F) male’s subventral lip (a – amphidial plate, white arrowhead – inner circle papillae; black arrowhead – cephalic double papilla). (Scale bars for A = 30 μm, B, C = 30 μm, D = 5 μm, E = 20 μm, F = 10 μm.)
Male
Lateral alae commencing from the nerve ring level extending posteriorly becoming indistinct anterior to ventral sucker. Testis forming a loop anterior to mid-body level then extends posteriorly to seminal vesicle. Two blind diverticula present, opening in the middle region of seminal vesicle (Figure 1A; Figure 5C). Vas deferens comparatively short, occupying one-fifth of reproductive system total length. Ventral sucker rounded, supported by prominent cuticular rim (Figure 4B). Small rounded swelling leading to an elongated vesicle with granular content present between the body of the sucker and posterior side of the rim. Prominent caudal alae present (Figure 4E). Genital papillae vary in size and shape but are always larger then somatic papillae. Two pairs of large pedunculate papillae situated at the level of ventral sucker and support the anterior end of caudal alae; three papillae pairs present at cloacal level: one preanal pair, one closely spaced adanal pair and one tiny postanal pair seated slightly laterally to the cloacal aperture; one large pedunculate sublateral pair supporting the posterior part of caudal alae; five pairs postcloacal: one large subventral pair just posterior to cloacal aperture, one sublateral pair, two subdorsal pairs and one subventral closest to tail tip (Figure 5; Figure 7A). Multiple somatic papillae present anterior to ventral sucker as well as pre and postcloacal area. Spicules are long, subequal, alate, tessellated along most of their length with spirally twisted distal tips (Figure 2D–G; Figure 4D; Figure 6B, D). Proximal ends of spicules are widened, forming well-defined heads slightly irregular in size and turned towards the ventral side of the body (Figure 2F; Figure 6A). On the second third of their length, the spicules bend ventrally. Gubernaculum developed as a thickening of the dorsal cloaca wall with lateral ventrally pointed processes flanking the spicules and probably closing above them. A small accessory structure – the ‘scutum’ – is near the cloacal opening (see Discussion). The scutum has the form of a bent plate attached to the dorsal wall of the cloaca with the free end directed dorsally (Figure 2C, E, F, G; Figure 4C; Figure 6E (black arrowhead). Tail short, conical, curved ventrally with pronounced cuticular striation and long distal tip.
SEM images of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972, male posterior end. (A) ventral view (white arrowheads indicate genital papillae); (B) ventral sucker, (C) cloaca area (white arrowhead indicates scutum); (D) spicule tip protruding through cloaca aperture, ventral view; (E) anteroventral view (white arrowhead indicates genital papillae). (Scale bars for A = 20 μm, B = 40 μm, C, D = 20 μm, E = 15 μm.) SEM, scanning electron microscopy.
Light microscope images of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972. (A) male head end, sublateral view; (B) female head end, sublateral view; (C) blind diverticula, opening in the middle region of seminal vesicle; (D) caudal ala supported by three pedunculate papillae (black arrowheads); (E, F) excretory pore in male, ventral and lateral views, correspondingly (black arrowhead indicate excretory pore orifice); (G) immature eggs surrounded with spermia; (H) mature egg with oval polar body marked with black arrowhead. (Scale bars for A = 25 μm, B = 20 μm, C = 200 μm, D-F = 25 μm, G, H = 50 μm.)
Light microscope images of males of Meteterakis striatura Oshmarin & Demshin, Reference Oshmarin and Demshin1972. (A-E) Meteterakis amamiensis Hasegawa, Reference Hasegawa1990 (F) and Meteterakis ishikawanae Hasegawa, Reference Hasegawa1987 (G). (A) Left spicule proximal end; (B) posterior body end; (C) cloaca region showing muscle cords braiding spicules (white arrowhead), gubernaculum (white arrow) and scutum (black arrowhead), lateral view; (D-F) cloaca region of M. striatura, M. amamiensis, and M. Ishikawanae, consequently, showing scutum (black arrowhead) and gubernaculum (white arrow), lateral view. (Scale bars for A, C, D = 25 μm, B = 200 μm, E-G = 10 μm.)
A maximum likelihood tree based on 28S rDNA of Meteterakis spp. Bootstrap values of 50% or higher are shown above the branches. Newly obtained sequences are indicated in bold.
Female
Lateral alae starts soon after oral opening anterior to nerve ring level and is continuous posteriorly, becoming indistinct at phasmid level close to tail tip. Vulva is slit-like without a flap (Figure 1E, F). Vagina vera is short, muscular, posteriorly directed, densely braided with muscular sphincter and bended ventrally before opening into the vulva. Vagina uterina long, posteriorly directed adjoining common uterus which divides posteriorly into two uteri filled with mature eggs (Figure 1D). Uteri run posteriorly joining oviducts close to anal level and then turn anteriorly joining ovaries placed anterior to vulva level (Figure 1B). Ovaries long, anteriorly directed, flexing posteriorly at basal bulb level, and ending at vulva level. Eggs oval with thick, smooth shells, embryonated at morula stage. Small oval polar body can be observed on one of the poles of the eggs (Figure 5G, H). Eggs, occupying the vagina uterina, are orientated by the polar body towards the vulva opening. Anal opening slit-like; rectum elongated, with thick cuticular lining (Figure 3B). Tail long, conical, gradually passing into cuticular tip.
Host and locality
Intestine of Geoemyda spengleri (Gmelin, 1789) (Geoemydae) collected from Guangxi Province of China and kept in captivity in Moscow Zoo, Russian Federation.
Deposited material
Single male and two females voucher specimens are deposited in the National Nematode Collection of New Zealand under the following numbers: 3500-3503. A male voucher specimen No. 14351 is deposited in the Centre of Parasitology, Institute of Ecology and Evolution, Russian Academy of Sciences, collection.
Remarks
Meteterakis striatura was discovered during a scientific expedition to Vietnam conducted by the Pacific Research Institute of Fisheries and Oceanography (now Russian Federal Research Institute of Fisheries and Oceanography) from December 1959 to December 1960 and then from April to September 1961. The host and parasite material were collected by P.G. Oshmarin, Yu.L. Mamaev and A.M. Parukhin. During the expedition, 10 specimens of tortoise Mauremys mutica (Cantor, 1842) (as Clemmys mutica Boettger, 1888) were collected from two localities: the vicinity of the Tam Dao village and nearby Haiphong City. Although the collection site for the nematode species’ host is not specified in the description, the collection date aligns with the collections in Tam Dao village. The original description does not provide deposition numbers of type slides or the location of the deposited material. Differential diagnosis given in the original description states that M. striatura can be distinguished from the other representatives by the morphology of the basal bulb, the presence of a striated cuticle on the ventral side of the tail, and lateral rows of large cells within the hypodermis. Due to the lack of a full differential diagnosis in the main description, we give it below in the present paper. Despite the poor taxonomic value of listed morphological peculiarities (see Discussion), such morphological characteristics as male and female body sizes, number of genital papillae in males, and general spicule morphology, illustrated in the main description, let us conclude that the species studied herein is conspecific with M. striatura.
Differential diagnosis
Meteterakis striatura is characterized by long alate spicule with a distinct tessellation in males and the absence of a vulval flap in females. Spicules exceeding 500 μm in length make M. striatura closer to the following congeners: M. amamiensis Hasegawa, Reference Hasegawa1990, M. aurangabadensis Deshmukh & Choudhari, 1980, M. formosensis Sata, Reference Sata2018, M. ishikawanae Hasegawa, Reference Hasegawa1987, M. japonica (Wilkie, Reference Wilkie1930), M. karvei Naidu & Thakare, 1981, M. lombokensis Purwaningsih, Dewi & Nugroho, Reference Purwaningsih, Dewi and Nugroho2016, M. longispiculata (Baylis, Reference Baylis1929), M. louisi Inglis, Reference Inglis1958, M. occidentalis Sata, Reference Sata2018, M. pursatensis Bursey, Goldberg & Grismer, 2019, M. singaporensis (Sandosham, 1953), M. vaucheri Adamson, Reference Adamson1986, M. wangi Zhang & Zhang, Reference Zhang and Zhang2011 and M. wonosoboensis Purwaningsih, Dewi & Hasegawa, Reference Purwaningsih, Dewi and Hasegawa2015. Males of M. striatura can be distinguished from M. louisi, M. singaporensis, M. vaucheri, and M. wangi by having smaller spicule size (678–688 μm vs 970–1100 μm, 740–960 μm, 1057–1242 μm and 740–930 μm, respectively). Discussed species has alate spicules which makes it different from M. aurangabadensis and M. longispiculata, which possess non-alate spicules. Meteterakis striatura varies significantly from M. amamiensis, M. formosensis, M. japonica, M. karvei, and M. occidentalis by having slender spicules with moderately developed heads. In contrast, in listed species, spicules are robust with funnel-shaped proximal ends. Compared with M. wonosoboensis, whose males also have slender alate spicules, M. striatura can be differentiated by the absence vs presence of vulvar flap in females. Meteterakis striatura is closest to M. lombokensis, M. pursatensisand M. ishikawanae in having close body dimensions in males and females, absence of vulva flap and general spicule morphology with well-developed ala and moderately developed proximal ends. However, all four species can be differentiated by the specific spicule shape. In M. lombokensis, proximal ends of spicules are strongly curved ventrally, making a C-shaped appearance in the lateral position, whereas in M. striatura spicules, distal ends are straighter. Meteterakis pursatensis, in contrast, has straighter proximal ends of spicules than observed in M. striatura. In addition, in M. pursatensis spicules, their distal third bends under 90 degrees towards the ventral body wall when smoothly bent in M. striatura. In its morphological and morphometric characteristics, M. striatura is undoubtedly closest to M. ishikawanae described from Odorrana ishikawae (Stejneger, 1901) (as Rana ishikawae [Stejneger, 1901]) of Okinawa Island, Japan (Hasegawa, Reference Hasegawa1987). Both species have very similar body measurements in males and females, spicule size and morphology, number and distribution of genital papillae in males, and females lack vulval flap. However, males of M. striatura can be differentiated by the narrower proximal parts of spicules blades bending in an S-shape, while in M. ishikawanae, proximal parts of spicules smoothly bend posteriorly to spicules heads’ which are less prominent than in M. striatura.
Molecular characterization
The 18S and 28S rDNA sequences obtained for three nematode specimens were identical. Molecular phylogenetic analysis of the 28S rDNA sequence (D2-D3 expansion segment, alignment length 735 bp) based on the newly obtained data and sequences available for the genus Meteterakis shows the presence of two well-separated clades with good support (Figure 7). Meteterakis striatura joins the same clade with M. japonica and M. ishikawanae while another clade is represented by M. occidentalis, M. formosensis, M. amamiensis and Meteterakis spp., the latter obtained from Plestiodon stimpsonii (Thompson, 1912) (from Okinawa, Ishigaki, and Mihara locations). The inter-specific relationships are resolved only for representatives of M. japonica. The level of nucleotide difference between M. striatura and M. japonica group is 6 bp, and 3 bp between the studied species and M. ishikawanae. Within the species M. japonica and M. ishikawanae, differences are 2 and 3 bp, respectively. The nucleotide differences between M. striatura and representatives of the other clade reach 13 and 17 bp (M. occidentalis and M. amamiensis, respectively), but within the clade do not exceed 22 bp (Meteterakis spp. and M. amamiensis). Comparison of data for 18S rDNA sequences (alignment length 845 bp) showed complete identity of sequences for M. striatura and M. japonica and 2 bp difference with M. amamiensis.
Discussion
The distinguishing characteristics of M. striatura, as outlined in the primary description by the authors, encompass several key features, including the shape of the basal bulb, notably the anterior portion resembling a truncated cone (see Figure 58, 2 after Oshmarin & Demshin, Reference Oshmarin and Demshin1972); the presence of striated cuticle on the ventral side of the tail, lateral rows of large cells within the hypodermis and a transverse fold situated on the ventral aspect of the body, observed immediately posterior to the lips. The present authors consider that the presence of the transverse fold and the unique structure of the basal bulb may be attributable to muscular contraction during specimen fixation. Some male specimens examined in the present study exhibited all described features, including protrusion of the cloacal region, as depicted in the original description; however, our molecular analyses conducted on three distinct individuals provide evidence supporting their conspecificity. Moreover, groups of 4–5 cells resembling the ones described by Oshmarin & Demshin (Reference Oshmarin and Demshin1972) were identified within the pseudocoelom in studied male specimens and probably represent pseudocoelomocytes.
Observation of the type material of M. amamiensis and M. ishikawanae, kindly provided by Meguro Parasitological Museum (Tokyo, Japan), confirmed the high morphological similarity between the latter species and studied herein M. striatura. Hasegawa (Reference Hasegawa1987) does not mention M. striatura in the differential diagnosis, although he gives a bibliographic reference for the latter species. Such morphological similarity probably gave rise to some suspicions about the co-specificity of these nematodes. The present study revealed some morphological differences in the shape of spicules and position of scutum between M. striatura and M. ishikawanae, given previously in the differential diagnosis. Based on Inglis’s (Reference Inglis1958) statement that spicule shape is of primary importance in distinguishing the species within Meteterakis and our molecular analysis that did not determine the relationships between the discussed species, we recognize M. striatura and M. ishikawanae as non-co-specific. Hasegawa (Reference Hasegawa1990) notes that he supposed M. ishikawanae to be specific to Rana ishikawae (Anura: Ranidae); however, later, it was discovered by his colleague in the tortoises Geoemyda japonica Fan, 1931 (given as Geoemyda spengleri japonica Fan, 1931) (Testudines: Geoemydidae) on Okinawa Island (Nakachi, personal communication; author’s note). We assume that the monophyletic origin of G. spengleri and G. japonica and the discovery of a fossil species, Geoemyda amamiensis from Tokunoshima Island, closely related to G. japonica, support Hasegawa’s earlier suggestion that Meteterakis spp. could be introduced to Okinawa Island from continental China by some reptiles (Hasegawa, Reference Hasegawa1987; Reference Hasegawa1990; Yasukawa et al., Reference Yasukawa, Hirayama and Hikida2001; Takahashi et al., Reference Takahashi, Kato and Ota2007; Yasukawa & Ota, Reference Yasukawa and Ota2008).
According to Inglis (Reference Inglis1958), disagreements regarding the position and independence of the genus Meteterakis were caused by an underestimation of the importance of such morphological characters as gubernacular mass and the number of male caudal papillae. The term ‘gubernacular mass’, which later became confusing and ignored by some authors, was proposed by Inglis (Reference Inglis1958) to describe the cuticularized structure located in the cloacal region and surrounding spicules. According to Inglis (Reference Inglis1958), the gubernacular mass is a characteristic feature for the present genus which can be more or less developed in all its representatives; this mass is a thickening of the cuticular lining of the walls of the cloaca more developed on the dorsal and ventral side and less pronounced laterally. Inglis (Reference Inglis1958) emphasizes that the true dimensions of a given structure are often difficult to determine and highlights it in his illustrations for the genus revision with a dotted line. According to Inglis (Reference Inglis1958), authors often mistakenly determine its size and shape; thus, in the description of Spinicauda bufonis (Yamaguti, Reference Yamaguti1935), the author depicts only the ventral part of this structure, denoting it as a ‘gub’ (apparently implying the gubernaculum). In the technique section of his article, Inglis (Reference Inglis1958) states that the confusion that takes place in the literature associated with the presence or absence of a gubernaculum in Meterakis representatives initially arose because of the incorrect application of clearing techniques when specimens had been cleared with lactophenol, and further over-cleared with creosote, causing the internal cuticular structures including spicules to become indistinguishable – especially in small representatives of the genus. In further descriptions and revisions of the Meteterakis genus, the term ‘gubernacular mass’ was neither frequently used (Cruz & Ching, 1975; Junker et al., Reference Junker, Mariaux, Measey and Mutafchiev2015; Purwaningsih et al., Reference Purwaningsih, Dewi and Nugroho2016) nor discussed. However, some authors (Oshmarin & Demshin, Reference Oshmarin and Demshin1972; Hasegawa, Reference Hasegawa1987) highlighted the area at cloacal opening with a dotted line without further discussion. Oshmarin and Demshin (Reference Oshmarin and Demshin1972) in their description of M. striatura do not use the term ‘gubernacular mass’, although they highlight some dotted outlines in the illustration of the posterior end of the male, while drawing attention to another cuticular structure near the cloaca opening, suggesting that it may be a gubernaculum.
Observation of present material, including paratype specimens of M. amamiensis and M. ishikawanae, revealed the presence of massive sclerotization of the dorsal cloaca wall soon after the cloaca opening in the area of passage of the distal ends of spicules. It is tightly woven with muscles and hard to observe. Present sclerotization is approximately 100–110 μm in length, has a saddle shape when observed laterally, and has muscle attachment along its proximal edge. The distal end of the discussed structure has a swelling resting against the lower cloacal lip. In M. striatura, at the anterior part of the sclerotization, we observed lateral, ventrally pointed processes flanking the spicules and closing above them. In addition, in all studied representatives of the genus, we observed a small cuticularized structure immediately near the cloaca opening (Figure 2C, E, F) resembling a thin plate of subtriangular or hastate shape with the free edge directed deep into the cloaca. In M. striatura, this structure is very small, translucent, and poorly visible in a light microscope. However, as observed from our material, in some cases, it can protrude outward and is visible on SEM images (Figure 5D). The exact structure protruding outside the cloaca aperture was observed in M. amamiensis and M. ishikawanae (Figure 6F, G). An analysis of the literature has shown that this structure can also be observed in SEM images of M. japonica (see Figure 33 in Purwaningsih et al., Reference Purwaningsih, Dewi and Hasegawa2015), M. lombokensis (see Figure 2S in Purwaningsih et al., Reference Purwaningsih, Dewi and Nugroho2016), and M. wangi (Figure 2E in Zhang & Zhang, Reference Zhang and Zhang2011). In the illustrations for M. wangi there is an image of a partially damaged structure, which is designated as the gubernaculum (see Figure 1H in Zhang & Zhang, Reference Zhang and Zhang2011), but unfortunately it is not shown in association with the spicules in the drawing of the male posterior end, and it is difficult to identify its origin. Adamson (Reference Adamson1986) in the description of M. vaucheri has illustrated a small subtriangular detail of male genital cone (see Figure 1N in Adamson, Reference Adamson1986). We believe that same structure is marked as the gubernaculum in the following species: M. asansolensis (see Figures 1E and 1G in Sou, Reference Sou2019), M. guptai (Gupta & Naiyer, Reference Gupta and Naiyer1993), M. hurawensis (Bursey et al., 2017), M. japonica (in the original description by Wilkie [Reference Wilkie1930]), M. wonosoboensis (Fig. 8 in Purwaningsih et al., Reference Purwaningsih, Dewi and Hasegawa2015) and illustrated on the drawings for M. formosensis and M. occidentalis and marked as posterior cloacal lip (see Figure 2F, G; Figure 3F, G in Sata, Reference Sata2018).
A characteristic feature of the male reproductive system in nematodes is the presence of such cuticular structures as spicules, gubernaculum and telamon. Based on the definitions given by different authors (Lee, Reference Lee2010; Mehlhorn, Reference Mehlhorn and Mehlhorn2016) the gubernaculum is formed from a dorsal thickening of a dorsal wall of the spicular pouch. It is essentially a curved plate forming a groove in which the spicules can move. The term ‘telamon’ was proposed by Hall (Reference Hall1921) to describe a small cuticular structure found in males of Hyostrongylus rubidus (Hassall & Stiles, 1892). The telamon is developed from the ventral wall of the cloaca near its aperture. It extends laterally into the lateral walls and then anteriorly as flattened curved plates in the lateral walls of the cloaca. Both the gubernaculum and telamon appear to be protective structures developed to support the walls of the cloaca and its aperture from the passage of spicules; they also support and guide spicules when extruded. According to Hall (Reference Hall1921), the term ‘gubernaculum’ should be restricted to the longitudinal structure in the dorsal wall of the cloaca toward its anterior end, and the term ‘telamon’ should be used for the supporting structure of variable form near the cloacal aperture. Hall assumed the presence of telamon in some Oxyurida, Spiruroidea, and Filarioidea. However, the term is mainly used to describe the accessory cuticular structure in the Strongyloidea (Hall, Reference Hall1921; Lee, Reference Lee2010).
Based on these definitions and our observations, the term ‘gubernacular mass’ refers to the massive cuticular sclerotization of the dorsal wall of the cloaca, which corresponds to the definition of the gubernaculum. We consider that small cuticular formation observed near the cloaca opening is associated with the gubernaculum and most probably serves is an accessory piece of the latter. To avoid further confusion about the mentioned structure, we propose using the term ‘scutum’ from the Latin word meaning ‘shield’ relating to form and partially to function, namely the reflection/direction of spicule blades. Apparently, the scutum plays the function of a guiding organ and perhaps serves for anchoring with a female’s vulval flap during copulation. As observed, the scutum is better developed in M. amamiensis, possessing a well-developed vulval flap. In contrast, in M. striatura and M. ishikawanae, where females lack a vulval flap, the scutum is less pronounced. We believe this structure is present in all representatives of the genus but is often overlooked because it resembles the cloaca lip.
Another important morphological feature found in males of M. striatura and marked for M. amamiensis and M. ishikawanae (present study) and perhaps overlooked in other species is the presence of blind diverticula in the male reproductive system. Previously, a single blind diverticulum was noted only for M. vaucheri (Adamson, Reference Adamson1986). The purpose of such a morphological formation is not entirely clear; diverticula originate at the same level and are outgrowths of the wall of the seminal vesicle filled with sperm. The sizes of diverticula differ slightly from each other.
Adamson (Reference Adamson1986) pointed that such important morphological and taxonomic feature as number and disposition of caudal papillae in males of Meteterakis causes difficulties because of the presence of a large number of somatic papillae that can be easily confused with genital papillae; and also, with the fact that the number of genital papillae can vary within a species. There is no doubt that the number of papillae is certainly an important diagnostic feature, and re-examination of type materials of previously described species is highly desirable, but this was not considered within the scope of present work. For this reason, in the diagnosis of the genus, we indicate the range within the change in the number of indicated genital papillae for species of the genus that are valid in our opinion.
Baker (Reference Baker1984) in the analysis of Meteterakinae and Spinicaudinae accepts the synonymy of M. longispiculata and Heterakis (Spinicauda) cophotis Baylis, Reference Baylis1936 proposed by Crusz and Sanmugasunderam (Reference Crusz and Sanmugasunderam1973) and lists 16 nominal species of Meteterakis genus including Meteterakis bufonis (Biswas & Chakravarty, 1963), as a new combination for Heterakis bufonis Biswas & Chakravarty, 1963 from B. melanostictus (Calcutta, India). The authors of the present study disagree with Baker’s (Reference Baker1984) assumptions regarding the inclusion of Heterakis bufonis Biswas & Charkavarty, 1963 as a representative of the genus Meteterakis due to the absence of the lateral alae in females, which is the strong diagnostic character of the genus. We place this species as species insertae sedis together with Africana bufonis Biswas & Chakravarty, 1963, as proposed by Baker (Reference Baker1984).
Meteterakis sinharajensis (Crusz & Ching, Reference Crusz and Ching1975) and M. lyriocephali (Crusz & Ching, Reference Crusz and Ching1975), which were referred to Meteterakis by Chabaud (Reference Chabaud, Anderson, Chabaud and Willmott1978), also raise some doubts about the validity of these species. Both species (see Crusz & Ching, Reference Crusz and Ching1975) have been described from the same host specimen of Lyriocephalus scutatus (Linnaeus, 1758) (Squamata: Agamidae) from Sri Lanka, where 3 males and 30 females were identified as representatives of Meteterakis spp. and remaining 33 males and 44 females were referred to a new genus named Cometeterakis because of distinctly unequal in size spicules. Crusz & Ching (Reference Crusz and Ching1975) also note that they found representatives of M. sinharajensis in Pseudotyphlops philippinus as well and give measurements that do not overlap either in the sizes of males and females, or the sizes of male spicules. In the description of C. lyriocephali, the authors of the species indicate a large variation in the sizes of the right and left spicules, as well as varying positions and numbers of sessile papillae. However, illustrations by Crusz and Ching (Reference Crusz and Ching1975) show that the number of large papillae also varies, suggesting the presence of more than two species in a single host. Given these inaccuracies, we place both species as species inquirendum.
Inglis (Reference Inglis1957b, Reference Inglis1958) points out the morphological isolation of two representatives of the genus: M. mabuyi and M. triaculeata. Both species are distinguished by the insufficient degree of development of the anterior cuticular flange of the lips; females of M. mabuyi lack the lateral alae (present morphological character is not mentioned in the description of M. triaculeata). Inglis (Reference Inglis1958) could not confirm the presence of the gubernacular mass in M. mabuyi since only a single female was observed; in males of M. triaculeata, gubernacular mass was not observed. Inglis (Reference Inglis1958) considered all three mentioned characters essential in determining the genus but left both species within Meteterakis due to the restricted amount of studied material. However, the situation with the ‘gubernacular mass’ has been clarified (present study), and the absence of the lateral alae, which plays an essential role in species identification (Sata, Reference Sata2019), makes the authors place both species as species inquirendae until the next morphological revision of the type material.
Based on the similarity of morphometric parameters of M. gambhiri (Gambhir et al., Reference Gambhir, Tarnita, Chinglenkhomba, Gyaneswori and Indrani2006) (described as M. bufonis and later renamed by Zhang & Zhang [Reference Zhang and Zhang2011]) and the type species M. govindi, we place M. gambhiri as a junior synonym of the latter species. Also, special attention deserves to be given to M. crombiei, described from Sphenomorphus jobiensis (Meyer, 1874) (Sauria: Scincidae) from Fergusson Island, Papua New Guinea (Bursey et al., Reference Bursey, Goldberg and and Kraus2005). This is the only species of the genus for which eggs with a tuberculate shell surface are described, while in all other congeners the eggs have a smooth shell. The absence of a differential diagnosis in the description of the species, as well as a special morphology that matches the description of eggs in some representatives of the genus Spinicauda, made by the same authors force us to place this species to the species inquirenda until clarifying the diagnosis and morphological features of the eggs.
Unfortunately, no reliable data regarding the life cycle of Meteterakis is available; Sata (Reference Sata2019) inclines to Anderson’s (Reference Anderson2000) assumption that they may be similar to Spinicaudinae when infection of the definitive host occurs through the eggs containing a third-stage larva. The high dispersion of the present parasitic group and lack of host specificity may also suggest the involvement of paratenic invertebrate hosts, such as lumbricids or terrestrial mollusks, which tend to be a common food source for most of Meteterakis hosts.
The molecular research of Meteterakis spp. performed by Sata (Reference Sata2018) for the East Asian islands, has revealed the presence of two genetically diverse lineages within the studied genus, namely J and A groups, which demonstrate different patterns of speciation. Thus, diversification within the J group (includes M. japonica and M. ishikawanae) correlates with the host biogeography and suggests co-divergence with vicariance event of the host fauna. However, in the A group (which includes M. amamiensis, M. occidentalis, M. formosensis and two undescribed species from Iriomotejima and Ishigakijima Islands, respectively) phylogenetic diversity does not correspond to the host biogeography and implies diversification by repeated colonization. Based on an analysis of data performed by Sata (Reference Sata2018), he concluded that any continental lineage close to the J group (including M. japonica and M. ishikawana) was expected to be relictual or absent. Our data refute the present assumption since the species studied herein and reported by the authors in this current paper species join Sata’s J group in all analyses conducted. However, the relationships within the group remain unresolved. All Meteterakis species studied using molecular techniques have very similar spicule morphology and, according to the division of Junker (Junker et al., Reference Junker, Mariaux, Measey and Mutafchiev2015), belong to the same group: a group whose spicules are equal and intermediate in length. However, there is a significant difference in the spicules morphology: in the A group, spicules are robust with funnel-shaped proximal ends, while in the J group, spicules are more slender with moderately developed heads. In addition, lateral alae are better developed in the J group, while narrow or absent in A-group species.
More sampling of Meteterakis spp. from the mainland belonging to different morphological groups differing in the spicules’ morphology would be desirable to obtain a more reliable picture of the genus phylogeny.
Acknowledgements
The present research was conducted within the framework of the Biological Diversity program No. 0109-2018-0075, ‘Ecology and diversity of parasitic organisms’. The authors are very grateful to Dr. Toshiaki Kuramochi and Dr. Hiroshi Saito from Meguro Parasitological Museum (Tokyo, Japan) and National Museum of Science and Nature (Amakubo, Japan), respectively, for providing the paratype material for M. amamiensis and M. ishikawana for comparative purposes. The authors also thank Dr. Devanshu Gupta, Officer-in-Charge of Coleoptera Section and Publication Division in Zoological Survey of India (Kolkata, India), Jamie Blackman, Fernandes Opook and Tithi Gandi from Manaaki Whenua Landcare Research (Auckland, New Zealand) for their help with finding the rare literature sources and Professor Alexey V. Tchesunov from Moscow State University (Moscow, Russia) for providing of nematode material.
Financial support
The Ministry of Business, Innovation and Employment (MBIE) in New Zealand partially funded the research through Strategic Science Investment Funding (SSIF) for Nationally Significant Collections and Databases.
Competing interest
None
Ethical standard
Not applicable
