Description of Pseudocapillaria (Discocapillaria) trilobularis n. sp. (Capillariidae) and redescription of Heliconema alatum (Majumdar, 1965) (Physalopteridae), two nematodes parasitising synbranchiform fishes in India

One new and one already known species of nematodes are described based on specimens collected from synbranchiform fishes of the River Ganga, India, in 2021 and 2022. Pseudocapillaria (Discocapillaria) trilobularisn. sp. (Capillariidae) from the intestine of cuchia Monopterus cuchia (Hamilton) (Synbranchidae) is mainly characterised by the presence of the large ventral postcloacal lobe in the male, the spicule length 351 µm, eggs size 60–69 × 30–36 µm (with protruding polar plugs) and by the body length (male 10.50 mm, females 11.02–12.44 mm). It is the fourth species of this genus recorded from fishes in India. The species Heliconema alatum (Majumdar, 1965) (Physalopteridae) is resurrected. This nematode is redescribed from specimens collected from the intestine of zig-zag eel Mastacembelus armatus (Lacepède) (Mastacembelidae). The examination by SEM revealed some previously not reported morphological features in this nematode species, e.g., the presence of cephalic papillae or a lateroterminal depression and two inner flat dorsoventral teeth on each pseudolabium. Based on these findings, H. monopteri Moravec, Chaudhary & Singh, 2019 is considered a junior synonym of H. alatum.


Introduction
As recently pointed out by Moravec et al. (2022), regardless of the large number of mostly taxonomic and faunistic papers treating freshwater fish nematodes in South Asian countries including India (Sood 2017), knowledge of the real fauna of these parasites in this region is still scarce. This unsatisfactory situation is not only due to the fact that many species of fish nematodes in this region were poorly described or incorrectly identified, but also because there are very many unsolved taxonomic problems in the respective nematode groups.
During parasitological examinations of some freshwater fishes in district Muzaffarnagar and Bijnor of Uttar Pradesh, India carried out in 2021 and 2022, adult nematode specimens were collected from the intestines of two species of synbranchiform fishes (Synbranchiformes), the cuchia Monopterus cuchia (Hamilton) (Synbranchidae) and the zig-zag eel Mastacembelus armatus (Lacepède) (Mastacembelidae). A closer examination of their morphology with the use of both light (LM) and scanning electron microscopy (SEM) showed that they belong to one new and one known but insufficiently described species. Results of their study are presented below. The results of molecular studies on these species will be published later.

Materials and methods
Fish were caught from district Muzaffarnagar (29.4727°N, 77.7085°E) and Bijnor (29.3833°N, 79.1833°E), Uttar Pradesh, India. The live nematodes obtained were washed in physiological saline and then fixed in hot 70% ethanol. For LM examination, they were cleared with glycerine. Drawings were made with the aid of a Zeiss microscope drawing attachment. Specimens used for SEM examination were postfixed in 1% osmium tetroxide (in phosphate buffer), dehydrated through a graded acetone series, critical-point-dried and sputtercoated with gold; they were examined using a JEOL JSM-7401F scanning electron microscope at an accelerating voltage of 4 kV (GB low mode). All measurements are in micrometres unless otherwise indicated. The fish nomenclature follows FishBase (Froese & Pauly, 2022 Etymology: The specific name trilobularis (= threelobular) is a Latin adjective and it relates to the threelobed appearance of the male caudal end of this nematode.
Male [Based on 1 specimen, holotype] Length of body 10.50 mm, maximum width 57. Width of lateral bacillary bands at region of posterior end of oesophagus 24. Length of entire oesophagus 5.48 mm, representing 52% of body length. Length of muscular oesophagus 246, of stichosome 5.39 mm; number of stichocytes about 35. Nerve ring situated 90 from anterior extremity. Seminal vesicle elongate-oval, 96 long, 36 wide (Fig. 1F). Spicular canal absent. Spicule well sclerotized, smooth, 351 long (Fig. 1F). Proximal end of spicule markedly expanded, 15 wide, without lobular rim; width of middle part of spicule 6; distal end narrowed, rounded, 3 wide (Fig. 1J). Spicular sheath without spines; contracted inner surface of withdrawn sheath appearing as spirally coiled thread surrounding anterior half of spicule (Fig. 1F). Posterior end of body obtuse, provided with 2 large, round ventrolateral lobes 6 long and 9 wide in lateral view; posterior lip of cloaca elevated, forming conspicuously large ventral lobe subdivided by median, longitudinal slit-like depression into 2 lateral parts; each part probably provided with indistinct papilla (Figs. 1E, K, 2A-C). Width of caudal end in lateral view 27, length of tail 12.
Specimens of the present new species are much larger than those of P. margolisi (body length of male 10.50 mm vs 0.99-3.16 mm, of female 11.02-12.44 mm vs 3.38-6.83 mm) and their stichocytes are usually more numerous (35-45 vs 24-37). The spicule of P. trilobularis n. sp. is distinctly longer than that of P. margolisi (351 lm vs 85-295 lm) and its proximal end is smooth, without lobular rim (vs with distinct lobular rim). Moreover, the marked longitudinal depression on the male ventral lobe, which is present in P. trilobularis n. sp., was not observed in P. margolisi. However, it is necessary to remark that the latter species was studied only by LM, whereas details of the male caudal end of the new species were revealed by SEM. In our opinion, the above-mentioned morphometrical differences between these two forms, and also the fact that hosts of these nematodes belong to different fish orders (Synbranchiformes vs Cypriniformes), are sufficient to consider the specimens from M. argus to represent a new species.
In contrast to P. margolisi and P. trilobularis n. sp., both representatives of the subgenus Discocapillaria, all species belonging to other three subgenera of Pseudocapillaria have no ventral caudal lobe in the male. Many of them also differ from the new species in having the spicule distinctly shorter (less than 300 lm) or longer (exceeding 440 lm). Only one species of the subgenus Pseudocapillaria, P. tomentosa (Dujardin, 1843), and three species of the subgenus Ichthyocapillaria, P. nannupensis, P. novaecaledoniensis and P. salvelini (Polyansky, 1952), have the spicule length similar to that of P. trilobularis n. sp. However, P. tomentosa, a parasite mainly of Holarctic cypriniforms (Moravec, 2001a), differs from the new species in having the anterior end of spicule provided with a distinct lobular rim (vs lobular rim absent). In contrast to P. trilobularis n. sp., the two male ventrolateral caudal lobes of P. nannupensis, P. novaecaledoniae and P. salvelini are connected between each other by a short dorsal cuticular membrane (vs dorsal cuticular membrane absent). Moreover, P. nannupensis, a parasite of the Australian freshwater catfish (Hobbs & Hassan, 2010), has smaller eggs (48-55 9 21-26 lm vs 60-69 9 30-36 lm) with non-protruding polar plugs, whereas P. salvelini, a parasite mainly of Holarctic salmonids (Moravec, 2001a), is characterised by the lobular anterior end of spicule (vs nonlobular). Pseudocapillaria novaecaledoniensis, a parasite of the marine perciform fish in New Caledonia (Moravec & Justine, 2010), also differs in smaller eggs (48-54 9 21-24 lm) and stichocytes subdivided into transverse annuli (vs stichocytes without annuli).
Pseudocapillaria trilobularis n. sp. is the first Indian capillariid studied by SEM. Unfortunately, anterior body ends of the three specimens examined by this method proved not to be clean enough to make detailed observation of the cephalic structures. Nevertheless, it can be assumed that the general structure of the cephalic end of this nematode is similar to those in other capillariids, i.e., that there are present two small lateral lips bearing minute amphids and 12 cephalic papillae in two circles surrounding lips (e.g., Baruš et al., 1981;Moravec, 2001b Heliconema alatum (Majumdar, 1965) De, 1988 Syns.: Notopteroides alatae Majumdar, 1965;Pseudoproleptus armati Sahay, Sinha & Sadhu, 1970;Paraleptus komiyai Sood, 1970;Heliconema kherai Gupta & Duggal, 1989 . Buccal cavity short. Oesophagus divided into short, narrow anterior muscular portion and much longer, wide glandular portion. Nerve ring encircles muscular oesophagus approximately at its middle or somewhat posterior to it. Small simple deirids situated just anterior to level of nerve ring (Fig. 3A). Excretory pore slightly anterior to anterior end of glandular oesophagus (Fig. 3A). Tail of both sexes with rounded tip.

Remarks
This species was poorly described by Majumdar (1965) as Notopteroides alatae from specimens collected from the intestine of M. armatus at Culcutta, India. Later Johnston & Khera (1967) synonymised Notopteroides Chakravarty & Majumdar, 1962with Pseudoproleptus Khera, 1955, to which they had transferred this species, renaming it P. alatus. Irrespective of the ICZN, Sahay et al. (1970) renamed the same species as P. armati. However, only De (1988), based on the re-examination of the deposited paratype specimens of N. alatae, showed that this species belongs, in fact, to Heliconema Travassos, 1919 (Physalopteridae) and he synonymised it with H. longissimum (Ortlepp, 1922). However, as explained by , the species H. longissimum was inadequately described from snakes in Australia and this name should only be used for the type specimens originally studied by Ortlepp (1922), until this species is redescribed in detail based on a newly collected topotypic material and the validity of H. longissimum is confirmed. Therefore, the records of H. longissimum from freshwater or brackish-water fishes should be considered as misidentifications, including those from M. armatus in India by Ogden (1969) and De et al. (1978).
The latter authors and De (1988) considered Paraleptus komiyai Sood, 1970 from M. armatus in India to be identical with those studied by them and misidentified as H. longissimum.  designated the latter species as H. kherai Gupta & Duggal, 1989 (see Gupta & Duggal 1989), but, in accordance with the ICZN, H. alatum should be the valid name for this species, with H. kherai as its junior synonym.
Heliconema alatum was not previously studied by SEM and its most complete description based on LM was provided by De et al. (1978). However, some taxonomically important features, such as the cephalic papillae or the presence of a lateroterminal depression filled with irregularly lobular mass and two inner flat dorsoventral teeth on each pseudolabium, are reported here for the first time. The use of SEM made it also possible to establish the exact number and distribution of the male caudal papillae, which are otherwise difficult to study by LM, as well as the structure and numbers of ventral precloacal ridges, etc.
The measurements of the present nematodes agree well with those given for this species by De et al. (1978), except for the size of eggs, which were found to be somewhat larger (42-45 9 27 lm vs 37 9 22 lm) than reported by those authors (probably they measured less developed eggs).  described Heliconema monopteri Moravec, Chaudhary et Singh, 2019 from the synbranchiform fish Monopterus cuchia (Hamilton) in India, differentiating it from H. kherai (= a synonym of H. alatum) from M. armatus mainly by a distinctly postequatorial vulva and the presence of the pseudolabial lateroterminal depressions. However, as confirmed in this study, pseudolabial depressions are also present in nematodes from M. armatus and the position of their vulva is also distinctly postequatorial (at 63-67% of the body length). Since there are no morphological or biometrical differences between these species and because their hosts belong to the same fish order (Synbranchiformes), we consider H. monopteri a junior synonym of H. alatum.
There is another nematode species, Pseudoproleptus vestibulus Khera, 1955, reported from M. armatus in India, which is morphometrically very similar to H. alatum, differing from it mainly in the presence of a considerably longer vestibule in addition to some details in the structure of the cephalic end. Even though P. vestibulus belongs to a different nematode family (Cystidicolidae) than H. alatum (Physalopteridae), because of its inadequate descriptions (Khera, 1955;Soota & Sarkar, 1980), both these species were probably confused between each other in the past Indian helminthological literature. A detailed redescription of P. vestibulus, based on newly collected specimens from its type host (M. armatus), is highly needed, especially because this nematode is the type species of the genus.
Data Availability All samples used in this study have been deposited in the relevant curated, internationally recognised museum collection as outlined in this paper.

Declarations
Conflict of interest František Moravec is a member of the Editorial Board of Systematic Parasitology.
Ethical approval All applicable institutional, national and international guidelines for the care and use of animals were followed.