Nematode parasites of four species of Carangoides (Osteichthyes: Carangidae) in New Caledonian waters, with a description of Philometra dispar n. sp. (Philometridae)

Parasitological examination of marine perciform fishes belonging to four species of Carangoides, i.e. C. chrysophrys, C. dinema, C. fulvoguttatus and C. hedlandensis (Carangidae), from off New Caledonia revealed the presence of nematodes. The identification of carangids was confirmed by barcoding of the COI gene. The eight nematode species found were: Capillariidae gen. sp. (females), Cucullanus bulbosus (Lane, 1916) (male and females), Hysterothylacium sp. third-stage larvae, Raphidascaris (Ichthyascaris) sp. (female and larvae), Terranova sp. third-stage larvae, Philometra dispar n. sp. (male), Camallanus carangis Olsen, 1954 (females) and Johnstonmawsonia sp. (female). The new species P. dispar from the abdominal cavity of C. dinema is mainly characterised by the body length (5.14 mm), the lengths of markedly unequal spicules (163 and 96 μm) and gubernaculum (102 μm long) provided with a dorsal protuberance and a small, reflexed dorsal barb on its posterior portion. The finding of C. bulbosus represents the first record of this parasite a century after its discovery; the first study of this species by scanning electron microscopy (SEM) enabled detailed redescription. The finding of Johnstonmawsonia sp. in C. fulvoguttatus is the first record of a rhabdochonid nematode from a host belonging to the Carangidae family. Johnstonmawsonia africana Moravec & Puylaert, 1970 and J. campanae Puylaert, 1973 are transferred to Prosungulonema Roytman, 1963 as P. africanum (Moravec & Puylaert, 1970) comb. n. and P. campanae (Puylaert, 1973) n. comb.


Fish and their identification
Fish were purchased from the fish market in Nouméa, New Caledonia. Most fishes from the fishmarket were taken with mackerel nets within a few miles off Nouméa and were very fresh. All carangids were relatively young specimens, far from the maximum lengths reported for these species [67]. The following fish species were examined: Carangoides chrysophrys (Cuvier) (n = 3), C. dinema (n = 7), C. fulvoguttatus (Forsskål) (n = 10) and C. hedlandensis (Whitley) (n = 2) ( Table 1). Fish were identified by their morphology, and confirmation of identification, from photographs of specimens, was sought from experts in ichthyology (Ronald Fricke, Bernard Séret and Samuel Iglésias). Fish DNA was extracted from tissue samples using the NucleoSpin 96 Tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Sequences were obtained by amplification and sequencing the 5 0 region of the cytochrome oxidase subunit I (COI) mitochondrial gene using the primers FishF1 (5 0 -TCAA-CYAATCAYAAAATYGGCAC-3 0 ) and FishR1 (5 0 -TGAT-TYTTYGGYCACCCRGAAGT-3 0 ) [70]. Standard PCRs were carried out in a total volume of 20 lL, containing about 30 ng of DNA, 1 · 10· PCR buffer, 2 mM MgCl 2 , 200 lM mix dNTPs, 150 nM of each primer and 1 unit of Taq polymerase (Qiagen, Hilden, Germany). After an initial denaturation of 3 min at 95°C, the mitochondrial DNA was amplified through 39 cycles of 15 s at 95°C, 20 s at 48°C and 40 s at 72°C, with a terminal elongation for 5 min at 72°C. PCR products were purified and sequenced in both directions on a 3730xl DNA Analyser 96-capillary sequencer (Applied Biosystems, Waltham, MA, USA). Sequences were edited using CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA), compared with the GenBank database content using BLAST and deposited in GenBank under Accession Numbers KX712506-KX712510. Species identification was confirmed using the BOLD identification engine [59] and BLAST in GenBank. The fish nomenclature adopted follows FishBase [18].

Nematodes
Parasites were collected using a ''wash'' method [25]. The nematodes were fixed in hot 4% formalin or 70% ethanol. For light microscopic examination, they were cleared with glycerine. Drawings were made with the aid of a Zeiss drawing attachment. Specimens used for scanning electron microscopy were postfixed in 1% osmium tetroxide (in phosphate buffer), dehydrated through a graded acetone series, critical-point-dried and sputter-coated 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 indicated otherwise. The classification system of the Ascaridoidea adopted follows Keys to the Nematode Parasites of Vertebrates [1,19].

Molecular identification of fish
In all these cases, we consider that our morphological identifications were confirmed by high similarity (>99%) or identity (100%) to sequences registered under the same taxon name in databases.
Male: Not known.

Remarks
Available female specimens cannot be identified to generic level, because conspecific males are absent. These nematodes are characterised mainly by a relatively short body length and muscular oesophagus, stichocytes with distinct transverse annuli, a subterminal anus and especially by the shape, structure and size of eggs. To date, three species of capillariids have been reported from marine fishes of the family Carangidae: Pseudocapillaria carangi (Parukhin, 1971), P. decapteri (Luo, 2001) and Capillaria gracilis (Bellingham, 1840) [29,35]. Whereas C. gracilis is a parasite mainly of gadiform fishes and its record in the carangid Trachinotus carolinus (Linnaeus) may well be a misidentification, P. carangi and P. decapteri, both inadequately described, are reported only from members of Carangidae. Therefore, it is probable that our specimens also belong to Pseudocapillaria Freitas, 1959. Parukhin [51][52][53] reported P. carangi from 12 species of carangid fishes (including two Carangoides spp.) from the western part of the Indian Ocean (Monar Bay, Arabian Sea near Oman, Gulf of Aden, Red Sea, off southeastern coast of Africa), whereas P. decapteri was recorded from the North Pacific Ocean near Japan [29]. Although specimens of the present material may belong to one of these two species (which, however, may be identical to each other), their poor original descriptions and principally the absence of a male in our material do not allow us to assign the New Caledonian specimens to a species.
The finding of one female specimen of this species, reported as Capillariidae gen. sp. 3, in New Caledonian waters was recorded by Moravec & Justine [40]; however, the host reported as Carangoides oblongus (Cuvier) was in fact C. dinema [12].

Remarks
Lane [28] described a new cucullanid species, Bulbodacnitis bulbosa, from the bluefin trevally Caranx melampygus Cuvier off Sri Lanka and established the new genus Bulbodacnitis to accommodate it, because he considered the presence of the dorsal hemispherical cephalic elevation in this species to be of generic importance. However, Barreto [5,6] considered Bulbodacnitis Lane, 1916 a junior synonym of Cucullanus Müller, 1777, to which he transferred Lane's species. Nevertheless, Smedley [66] and Simon [65] described two new species of Bulbodacnitis from North American salmonids, while other authors [7,15,58,71] did not recognise Bulbodacnitis as an independent genus. Subsequently, Maggenti [31] re-erected Bulbodacnitis for the cucullanids with the oral aperture dorsally oblique to the longitudinal body axis (see also [21,48]). However, Petter [54] pointed out that this feature is not found in B. bulbosus, the type species of Bulbodacnitis, and, consequently, she retained Bulbodacnitis as a synonym of Cucullanus and established a new genus Truttaedacnitis Petter, 1974 for the species with the distinctly oblique oral aperture. According to Moravec [34], Truttaedacnitis should be considered as a subgenus of Cucullanus.
The present specimens from C. fulvoguttatus correspond, more or less, to the description of C. bulbosus, both these forms were collected from carangid fishes, and C. melampygus, the type host of C. bulbosus, also occurs in New Caledonian waters [18]. Therefore, the New Caledonian nematodes undoubtedly belong to C. bulbosus.
Cucullanus bulbosus has not been recorded since its description by Lane [28], making the New Caledonian specimens the first finding of this species after a century. The original description of Bulbodacnitis bulbosa (= C. bulbosus) is relatively good (a somewhat modified description, based on the original one, was published by Baylis [7]). The present study, including the first scanning electron microscopy (SEM) examination, confirmed some previously reported morphological features in this species, showed some new characters (presence of circumoral spines and ventral oblique muscle bands in the male) and provided more exact observations of the cephalic structures and male caudal papillae. The finding of C. bulbosus in C. fulvoguttatus from off New Caledonia represents new host and geographical records.  (Lacépède) (Serranidae), H. cenaticum (Bruce & Cannon, 1989) [39,43]. In addition, unidentified larvae of Hysterothylacium have been reported off New Caledonia from several fish species of the Balistidae, Clupeidae, Lethrinidae, Nemipteridae, Scombridae, Serranidae, Sphyraenidae and Trichiuridae [23,24,64].
The life cycles and larval morphogenesis of Hysterothylacium spp. remain mostly unknown, making species identification of the larvae of this genus from fishes, based on morphological features, impossible. Shamsi et al. [63,64] distinguished 14 morphotypes of larval Hysterothylacium spp. (types I-XIV) from marine fishes in Australian and New Caledonian waters, of which types VI, XIII and XIV were recorded from fishes from off New Caledonia [64]. However, it is necessary to note that the ''sinusoidal'' or ''serpengenous'' patterns of the intestine, reported to be characteristic of the larval types VI and XIII, were in fact the coils of the developing genital tract, as is evident from the respective microphotographs (Figs. 2A and 2C of Shamsi et al. [64]).

Remarks
All the above-mentioned forms are considered to represent one and the same species of Raphidascaris Railliet & Henry, 1915, which attains full maturity in Carangoides spp. The presence of characteristic, anteriorly united lateral alae in fourthstage larvae shows that this currently undescribed species belongs to the subgenus Ichthyascaris Wu, 1949.
To date, 10 species of Raphidascaris (Ichthyascaris) are known as parasites of marine fishes [72]. Of these, five species were reported from the South Pacific Ocean in the Australian region: R.  [13,39,41]. However, none of the Raphidascaris (Ichthyascaris) spp. has so far been described from fishes of the family Carangidae. Therefore, it can be assumed that the nematodes parasitising Carangoides spp. in New Caledonian waters belong to a new species.

Remarks
Larvae of this type, designated as Terranova type II, were already reported from off New Caledonia by Shamsi et al. [64], who had recorded them from fishes of the families Carangidae    . N-1118).
Etymology: The specific name dispar (= unequal, disparate) is a Latin adjective and relates to the characteristic feature of this species, i.e. unequally long spicules.

Remarks
To date, eight nominal species belonging to four philometrid genera (Buckleyella Rasheed, 1963 [21,38,42]. By the body length 5.1 mm, the present male resembles only that of P. selaris (5.3-5.5 mm), whereas the males of other three species are distinctly shorter (1.5-3.3 mm). Moreover, both P. dispar sp. n. and P. selaris possess a dorsal reflexed barb at the tip of the gubernaculum, which is absent in other species. However, the new species differs from P. selaris in having conspicuously unequal spicules (length ratio of spicules 1:1.28 vs. 1:1.03-1.04), a different shape and structure of the gubernaculum (presence vs. absence of a dorsal protuberance) and a more posterior location of the oesophageal cell nucleus; in addition, it was collected from a fish belonging to a different genus (Carangoides vs. Selar). Males of the remaining four philometrid species from carangids are not known and, consequently, cannot be compared with P. dispar; however, these species can be separated based on the different genus of their type host and their geographical distribution. The allocation of the new species to Philometra is provisional; present philometrid genera are mostly based on the morphology of gravid and subgravid females, whereas males of some genera (e.g. Caranginema, Philometra and Philometroides) are unidentifiable to genus [42].
By the very unequally long spicules, P. dispar n. sp. differs from all other philometrid species from marine fishes, for which males are known, except for Philometra katsuwoni Petter & Baudin-Laurencin, 1986 and P. gymnosardae Moravec, Lorber & Konečný, 2007, both gonad-infecting parasites of tuna fishes (Scombridae) in the Atlantic and Indian Oceans, respectively [16,45,55]. However, differences in the lengths of spicules of P. katsuwoni and P. gymnosardae are much more conspicuous as compared with that in P. dispar.
The authors are aware of the fact that this new species is being described from a single specimen, a procedure that cannot be generally recommended; however, in this case, the new species appears to be readily recognisable and, therefore, they consider it more reasonable and useful to give it a specific name rather than to report it only as Philometra sp.

Remarks
Only two ovigerous female specimens, 11.06 mm and 18.43 mm long, were collected. The tail tip of both of them bears three small caudal projections. Since the general morphology of these specimens corresponds to that of C. carangis, as redescribed by Moravec et al. [44], they are assigned to this species.
Moravec et al. [44] observed that the tail tip of young nongravid and small subgravid (ovigerous) females of C. carangis possess three small caudal projections, whereas these are totally absent from conspecific gravid (larvigerous) females. This is confirmed by the present study.
Voucher specimen: Not maintained (used for SEM).

Remarks
In having a hexagonal oral aperture, no pseudolabia and a long vestibule (stoma) forming a distinct funnel-shaped prostom at its anterior end, the single available female specimen evidently belongs to the thelazioid family Rhabdochonidae. At present, this family consists of 11 genera, of which Trichospirura Smith & Chitwood, 1967 contains several species parasitising tetrapod vertebrates (amphibians, reptiles and mammals) [2], whereas the nematodes belonging to all other genera are parasites of fishes [36].
Of these, representatives of three genera, Rhabdochona Railliet, 1916, Prosungulonema Roytman, 1963 and Beaninema Caspeta-Mandujano, , are parasites of freshwater fishes; whereas Rhabdochona contains more than 100 species (all intestinal parasites) distributed in all main zoogeographical regions [37], Prosungulonema and Beaninema are represented by a few species parasitic in the host's intestine, liver, gallbladder or swimbladder in eastern Asia, Africa and Mexico [17,36]. On the contrary, the remaining seven genera, Johnstonmawsonia Campana-Rouget, 1955, Hepatinema Rasheed, 1964 species (most of these genera are monotypic) that are parasites in the digestive tract and associated glands, bloodstream and body cavity of marine and brackish-water fishes in tropical and subtropical regions [3,33,45,47].
All species of Johnstonmawsonia were described to have no teeth in the prostom. The present specimen has no anterior prostomal teeth, but its prostom is provided with six minute, more posteriorly located denticles, which are visible only with the use of SEM. However, it should be remarked that none of the Johnstonmawsonia spp. has so far been studied by SEM (except for the poor quality SEM micrograph of the cephalic end of J. porichthydis [68]). Therefore, it is not currently clear whether the presence of small posterior prostomal denticles is a generic feature in Johnstonmawsonia or it is only a character of an apparently undescribed congeneric species parasitising Carangoides fulvoguttatus.
The presence of posteriorly located prostomal teeth, as found in the present specimen of Johnstonmawsonia, is not exceptional among rhabdochonids. Megachona chamelensis Mejía-Madrid & Pérez-Ponce de León, 2007, a parasite of intestinal caecae of Kyphosus ocyurus (Jordan & Gilbert) (Kyphosidae) off the Pacific coast of Mexico, possesses many large anterior prostomal teeth and numerous smaller teeth with an irregular arrangement located more posteriorly [33]. On the other hand, the anterior prostomal teeth are absent in Fellicola longispiculus Petter & Køie, 1993, a parasite of the gallbladder of Coryphaenoides rupestris Gunnerus (Macrouridae) from off the Faroes, North Atlantic, but its prostom is internally lined with six longitudinal thickenings (four lateral simple and two median bilobed) appearing anteriorly as small denticles [56]. In Beaninema nayaritense Caspeta-Mandujano, Moravec & Salgado-Maldonado, 2001, a parasite of the gallbladder of Cichlasoma beani (Jordan) (Cichlidae) in Mexico, the anterior prostomal teeth are absent, but there are six large conical teeth in the posterior half of the prostom [17]. Both