Two new skink-endoparasitic species of Meteterakis ( Nematoda , Heterakidae , Meteterakinae ) from East Asian islands

Here, two new nematodes of Meteterakis Karve, 1930 from Taiwan and the western Japanese Archipelago that are endoparasitic to scincid lizards are described. The Taiwanese Meteterakis formosensis sp. n. and the Japanese Meteterakis occidentalis sp. n. can be distinguished from other congeners by the following characteristics: spicules 437–537 μm in length in M. formosensis sp. n. and 359–538 μm in M. occidentalis sp. n.; spicules with narrow alae, funnel-shaped, proximal ends ventrally bent; prevulval flap well-developed; gubernaculum mass absent; preclocal sucker with diameter of 35–47 μm in M. formosensis sp. n. and of 32–36 μm in M. occidentalis sp. n.; 9–15 caudal papillae on both lateral sides in M. formosensis sp. n. and 10–14 in M. occidentalis sp. n.; and relatively narrow lateral alae, ending at region near proximal end of spicule in male or at region anterior to anus in female. Meteterakis formosensis sp. n. is distinguished from M. occidentalis sp. n. by possessing spicules with hyaline pointed distal ends and well-developed cuticular backing structures. The present study suggests that lateral alae can be used as diagnostic character among the Meteterakis species, and it revealed that meteterakine nematodes mature in the host’s small intestine and then migrate to the rectum to oviposit.


Introduction
Meteterakis Karve, 1930 is a parasitic nematode genus, which is specific to amphibians (frogs and caecilians) and reptiles (lizards and land turtles) (Baker 1984, 1987, Hasegawa and Asakawa 2004, Zhang and Zhang 2011, Junker et al. 2015), and currently consists of 27 species.They are distributed in South, Southeast and East Asian regions, as well as in Oceania and Sao Tome Island, which is in the Gulf of Guinea (Baker 1984, 1987, Junker et al. 2015).Four species of Meteterakis have been recorded from the East Asian islands, which consist of the islands from the Japanese Archipelago to Taiwan: M. japonica (Wilkie, 1930), inhabiting the eastern Japanese Archipelago and Shimokoshikijima Island, an islet west off Kyushu, western Japan; M. amamiensis Hasegawa, 1990, indigenous to the western Japanese Archipelago and northern the Ryukyu Archipelago (Kodakarajima and Amamioshima Islands); M. ishikawanae Hasegawa, 1987, described from the Okinawan Islands; and the Burmese M. govindi Karve, 1930, which is the type species of the genus, from southern Taiwan (Karve 1930, Wilkie 1930, Yamaguti 1935, 1941, Hasegawa 1987, 1990, 1992, Telford Jr 1997, Goldberg and Bursey 2002, Bursey et al. 2005, Norval et al. 2014, Sata 2015, 2018).Although M. japonica was once reported from Miyakojima Island in the southern Ryukyu Archipelago (Hasegawa 1984), the individuals from the islet can be distinguished from the "true" M. japonica by the absence of a gubernaculum (M.japonica possesses a gubernaculum; Wilkie 1930, Inglis 1958).
A molecular phylogenetic study revealed that the East-Asian insular Meteterakis nematodes are divided into two major clades (Sata 2018).One phylogroup contains M. japonica and M. ishikawanae, while the other major clade contains M. amamiensis and three distinct unidentified nematodes from Ishigakijima and Iriomotejima islands in the southern Ryukyu Archipelago and Taiwan.The study also highlights the deep-genetic divergence between the M. amamiensis populations inhabiting Kodakarajima and Amamioshima (type locality) Islands and those distributed in the western Japanese Archipelago (Sata 2018).
Clarifying the systematic accounts of the aforementioned unidentified species will lead to a better understanding of the species diversity and evolutionary history of Meteterakis parasites inhabiting the East Asian islands.In the present study, therefore, the taxonomic states of the unidentified Taiwanese species and the M. amamiensis populations in the western Japanese Archipelago are investigated, and each is described as a new species.

Methods
The Meteterakis specimens examined in this study were obtained from the scincid lizard hosts, Plestiodon chinensis (Gray, 1838) and Plestiodon japonicus (Peters, 1864), which were collected from Taiwan and the western Japanese Archipelago (Fig. 1), respectively.The host lizard specimens were identified based on Okamoto and Hikida (2012) and Kurita et al. (2017).Hosts were collected and handled in accordance with the Regulations of Animal Experimentations at Kyoto University (approval numbers: H24014 and H2711).All captured lizards were euthanized by an injection of sodium pentobarbital.The body cavity of each specimen was dissected by a longitudinal incision, and then the digestive tract was removed.The excised organs were dissected longitudinally, and the lumens were investigated.The obtained nematode individuals were fixed with a hot 5% solution of glycerin in 70% ethyl alcohol.To clear the nematode specimens, they were placed in 50% solution of glycerin in 70% ethyl alcohol, then incubated 2-3 days at 60 °C to gradually evaporate the ethyl alcohol.The cleared specimens were observed with a light microscope (OLYMPUS BX53).The measurements in males were given for a holotype, followed by the range of paratypes in parentheses; for females, averages were provided, followed by the range of the paratypes in parentheses.All measurements were described in micrometers (μm) unless otherwise stated.Both the nematode and reptile specimens examined in this study have been deposited in the Zoological Collection of Kyoto University (KUZ).
For comparison, the following Meteterakis specimens deposited in the KUZ collection were examined: M. amamiensis (sensu Sata 2015): KUZ Z2015 from Yakushima Town, Kagoshima Prefecture, Japan (Yakushima Island), or to nerve ring or front end of nerve ring in both sexes and ending at region near proximal end of spicule in male (never reaching region of preclocal sucker) or at region anterior to anus in female.Prevulval flap present and well developed in female.Gubernacular mass absent.Spicules with thin alae, funnel-shaped proximal ends, hyaline tips, and both proximal and distal ends bent ventrally.Right spicule, 437-510 long; left spicule 457-537 long.Each spicule with thick and long cuticular backing structures, not covered by cuticular pouch.Caudal papillae present in male, 8-13 (N=4) pairs with additional papillae: 12-15 (N=4) on right side; 9-15 (N=5) on left side.
Etymology.The specific name is an adjective, derived from the old name for Taiwan, which is the type locality of the new species.
Description.General.Body short and relatively stout with tapered extremities.Cephalic end with 3 lips, each lip with 2 minute apical papillae.Dorsal lip with a pair of cephalic papillae (each papilla with 2 minute papillae); each subventral lip with single papilla (each papilla with 2 minute papillae), 1 amphid and 1 smaller papillae.Flanges in inner edge of each lip unobservable.Esophagus comprise of pharynx, cylindrical portion and bulb.Bulb bearing three valves.lateral alae commencing from region anterior to nerve ring or front end of nerve ring in both sexes and ending at region near proximal end of spicule in male (never reaching region of preclocal sucker) or at region anterior to anus in female.
In addition to the spicule length, the new species is distinguishable from the eight congeners by the following characteristics of spicules: proximal end wide, funnel-shaped and ventrally bent vs. proximal end slightly widened in M. ishikawanae (Hasegawa 1987) and M. wonosoboensis Purwaningshi, 2015 or vs. proximal end straight in M. guptai Gupta &Naiyer, 1993 andM. triaculeata (Kreis, 1933) (Inglis 1958) (Wilkie 1930, Inglis 1958, Bursey et al. 2017).M. formosensis sp.n. bears narrow lateral alae ending at the region near the proximal end of the spicule in the male or at the region anterior to the anus in the female.Thus, this new species differs from M. amamiensis, which is diagnosed by possession of the wider lateral alae ending at the preclocal region in the male, and at the region near the posterior end in the female (Hasegawa 1990).
Morphologically, M. formosensis sp.n. most resembles M. sp.n., which is described below, but it differs from the latter species by the female having a relatively stout body (body length/body wide: 25.8-27.8 in M. formosensis sp.n. vs. 31.8-41.2 in M. occidentalis sp.n.), relatively longer tail length in the female (body length/tail length: 10.7-10.9 in M. formosensis sp.n. vs. 13.6-17.8 in M. occidentalis sp.n.), spicules with hyaline tips (lacking in M. occidentalis sp.n.) and well-developed backing structures for spicules (undeveloped in M. occidentalis sp.n.).
Additional material.To reveal the geographic range of M. occidentalis sp.n., the following specimens deposited in the KUZ collection were examined: M. amamiensis (sensu Sata 2015): KUZ Z2014 from Kumamoto City, Kumamoto Prefecture, Japan (site 3 in Fig. 1), and KUZ Z658 and Z2022 from Kagoshima City, Kagoshima Prefecture, Japan (site 4 in Fig. 1).
Diagnosis.Short and slender body, with narrow lateral and caudal alae; lateral alae commencing from region anterior to nerve ring in both sexes and ending at region near proximal end of spicule (never reaching region of preclocal sucker) in male or at region anterior to anus in female.Prevulval flap well developed in female.Etymology.The specific name is a Latin adjective in the nominative singular, occidentalis (western), referring to its distribution in the western Japanese Archipelago.
Notes on the life cycle.Most Meteterakis nematodes collected from recta were adult individuals.Several larval nematodes were collected from the small intestines of the host individuals, which were inhabited by M. occidentalis sp.n., and a small number of ensheathed Meteterakis were also found from there.The rate of females having eggs in hosts' small intestines and recta were 36.2%(21/58) and 72.5% (50/69), respectively.
Comparisons.M. occidentalis sp.n. differs from M. formosensis sp.n. as discussed above.Because most of the morphological characteristics of M. occidentalis sp.n. are concordant with those of M. formosensis sp.n., this new species can be distinguished from the other congeners by the features mentioned in comparisons with M. formosensis sp.n. and the other Meteterakis species.Therefor, M. occidentalis sp.n. can receive the taxonomic status of a distinct species within the genus.

Discussion
Heterakidae has been classified into three subfamilies, Heterakinae, Spinicaudinae, and Meteterakinae (Inglis 1957).While the life cycles of heterakine and spinicaudine nematodes have been well documented, the life cycle of meteterakine species remains unknown (Anderson 2000).During the dissection and examination of host reptile specimens, larval nematodes were collected from the small intestines of P. japonicus.Because those larvae often co-occurred with adult worms of M. occidentalis sp.n. and/or ensheathed Meteterakis nematodes in the host materials, they are likely to be M. occidentalis sp.n. larval individuals.
Most of the M. occidentalis sp.n. specimens of both sexes collected from the recta were at the adult stage.Moreover, mature female of M. occidentalis sp.n. bearing eggs occurred less frequently in the hosts' small intestines than in the recta, suggesting that the larvae of this species may mature in the small intestine and then migrate to the rectum to oviposit.A similar life cycle has been recorded for two spinicaudine species that are parasites in the rectum of Malagasy chameleons (Anderson 2000).The present results provide new insights into the life cycle of the meteterakine nematodes and indicates that their life cycles may resemble those of spinicaudine species.
The spicule length, which has been regarded as a useful taxonomic character of the genus Meteterakis (e.g.Junker et al. 2015), exhibits certain intraspecific variations.Moreover, the characteristic of spicule length sometimes overlaps with those of other congeners.In contrast, the present study revealed an obvious morphological difference between the two morphologically similar species, M. occidentalis sp.n. and M. amamiensis, in the width and ending positions of the lateral alae.Because the characteristics of the lateral alae in M. occidentalis sp.n. showed much less intraspecific variation, lateral alae can be used as a diagnostic character among the Meteterakis species.The combination of the lateral alae and other traditional taxonomic character within this genus may help discriminate the species of Meteterakis.However, Meteterakis species, which were described previously, sometimes lack morphological descriptions of their lateral alae (e.g.Inglis 1958, Gambhir et al. 2006).Therefor, the characteristics of their lateral alae should be revisited by future taxonomic revisions.Additionally, it is preferable that future descriptive studies of new Meteterakis species contain the characteristics of lateral alae.
Because a previous phylogenetic study (Sata 2018) did not include "M.amamiensis" (sensu Sata 2015) specimens from Kumamoto City, Kumamoto Prefecture, Japan (Kyushu) and Yakushima Town, Kagoshima Prefecture (Yakushima Island), Japan, their taxonomical accounts remained unclear.The present morphological observations revealed that the Meteterakis specimens from Kumamoto Prefecture, and Yakushima Island are M. occidentalis sp.n., and M. amamiensis, respectively, based on the characteristics of their lateral alae.Although the herpetofauna of Yakushima Island is closely related to that of Kyushu, and their compositions have genetically diverged deeply from each congeneric species on the Amamioshima Islands (Tanaka-Ueno et al. 1998, Brandley et al. 2012), M. amamiensis is indigenous to Yakushima and Amamioshima Islands.Thus, the geographic range of this nematode species would be obviously discordant with the host biogeography.
This discordant pattern between the endoparasites and their host species was indicated by the two unidentified Meteterakis species on Ishigakijima and Iriomotejima Islands (Sata 2018).Therefore, the range of each Meteterakis species cannot always be predicted by the host's distribution pattern.Thus, the species diversity of Meteterakis should be revealed by geographically exhaustive faunal and taxonomic surveys.Moreover, the taxonomic account of the unidentified Meteterakis on Ishigakijima and Iriomotejima Islands should be clarified by a future study using appropriate specimens of the species.

A, B, F, G), paratypes (KUZ Z1781: D, J; KUZ Z1782: E; KUZ Z1994: H, I; KUZ Z1995: C). A anterior
region, lateral view; B pharynx, lateral view; C anterior end, apical view; D vulvar area of female, lateral view; E caudal region of female, lateral view; F caudal region of male, lateral view; G caudal papillae arrangement of male, lateral view; H spicule; I accessory of spicule; J egg.
Inglis, 1958., 2015s.roughsurface in M. saotomensisJunker et al., 2015; and spicule alae narrow vs. wider in M. baylisiInglis, 1958,  M. crombiei Bursey et al., 2005and M. sinharajensis Crusz & Ching, 1975.Meteterakis formosensis sp.n. is distinguished from M. lombokensis Purwaningshi et al., 2016 by the presence of a well-developed prevulval flap in the female.Additionally, this species possesses a 35-47 μm (diameter) preclocal sucker and elliptically-shaped eggs.These characteristics can be used to discriminate this new species from M. andamanensis Soota & Chaturvedi, 1972, which has a 55-66 μm (diameter) preclocal sucker and spherical-shaped eggs.The number of caudal papillae (9-15) on both lateral sides of the new species can distinguish it from M. paucipapillosa Wang, 1980 because the latter possesses only 6 caudal papillae on both lateral sides.The new taxon is clearly distinguishable form M. japonica and M. hurawensis Bursey et al., 2017 by the absence of a gubernacular mass