New records of spirurid nematodes (Nematoda, Spirurida, Guyanemidae, Philometridae & Cystidicolidae) from marine fishes off New Caledonia, with redescriptions of two species and erection of Ichthyofilaroides n. gen.

Recent examinations of spirurid nematodes (Spirurida) from deep-sea or coral reef marine fishes off New Caledonia, collected in the years 2006–2009, revealed the presence of the following five species: Ichthyofilaroides novaecaledoniensis (Moravec et Justine, 2009) n. gen., n. comb. (transferred from Ichthyofilaria Yamaguti, 1935) (females) (Guyanemidae) from the deep-sea fish Hoplichthys citrinus (Hoplichthyidae, Scorpaeniformes), Philometra sp. (male fourth-stage larva and mature female) (Philometridae) from Epinephelus maculatus (Serranidae, Perciformes), Ascarophis (Dentiascarophis) adioryx Machida, 1981 (female) (Cystidicolidae) from Sargocentron spiniferum (Holocentridae, Beryciformes), Ascarophis (Ascarophis) nasonis Machida, 1981 (males and females) from Naso lituratus and N. unicornis (Acanthuridae, Perciformes), and Ascarophisnema tridentatum Moravec et Justine, 2010 (female) from Gymnocranius grandoculis (Lethrinidae, Perciformes). Two species, I. novaecaledoniensis and A. nasonis, are redescribed based on light microscopical (LM) and scanning electron microscopical (SEM) examinations, the latter used in these species for the first time. Morphological data on the specimen of A. tridentatum from the new host species are provided. Philometra sp. (from E. maculatus) most probably represents a new gonad-infecting species of this genus. The newly established genus Ichthyofilaroides n. gen. is characterized mainly by the presence of a small buccal capsule and by the number and distribution of cephalic papillae in the female; it is the sixth genus in the Guyanemidae.

Recent examinations of spirurid nematodes collected by J.-L. Justine and his students in marine fishes from off New Caledonia in the years 2006-2009 revealed the presence of five species of these parasites, two of which were previously insufficiently well described and two are newly recorded in New Caledonian waters. Results of this study are presented herein.

Materials and methods
Fish were caught off New Caledonia by various means; fish from the deep-sea campaign were frozen-thawed. We generally used the "wash" method [18]. The nematodes for morphological studies were fixed in hot 4% formalin or 70% ethanol. For light microscopical examination (LM), they were cleared with glycerine. Drawings were made with the aid of a ZEISS microscope drawing attachment. Specimens used for scanning electron microscopical (SEM) examination 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 otherwise indicated. The fish nomenclature adopted follows FishBase [11].

Results
Family Guyanemidae Petter, 1974 Ichthyofilaroides n. gen. urn:lsid:zoobank.org:act:6E8F1B66-B534-4320-805F-5DCD273A8FFF Diagnosis: Dracunculoidea, Guyanemidae, Travassosneminae. Body of female cylindrical, with narrowed anterior and posterior ends; cephalic end rounded, posterior end pointed. Cuticle smooth. Oral aperture circular, surrounded by four small submedian cephalic papillae arranged in two circles and by pair of lateral amphids. Small but distinct, simple buccal capsule present. Oesophagus divided into anterior muscular and short posterior glandular portions; latter with long posteriorly oriented appendix. Intestine ending posteriorly in ligament attached to body wall. Amphidelphic. Posterior ovary much larger than anterior one, extending almost to anterior end of tail. Uterus continuous, filled with larvae. Vulva functional, situated in posterior half of body. Male not known. Parasites of musculature, surface of visceral organs and body cavity of marine fishes.

Remarks
Moravec and Justine [33] described the dracunculoid Ichthyofilaria novaecaledoniensis Moravec et Justine, 2009 from a single subgravid female obtained from the flesh near gills of H. citrinus off New Caledonia, which was studied solely by LM. Since the available specimen was fairly damaged, some of its important morphological features were given inaccurately, as visible from the present study, or were not observed at all. The four newly collected specimens (all gravid females) of this species made it possible to redescribe it based on both LM and SEM examinations.
In contrast to the original description, the present study has revealed the presence of a small, but distinct buccal capsule in this species, which indicates a different generic appurtenance of this nematode. In addition, the shape of the oral aperture, the number and arrangement of cephalic papillae and the presence of amphids and the functional vulva in this nematode are reported for the first time. The present study has also shown that the posterior appendix of the glandular oesophagus is much longer as compared with the original description.
However, as revealed in this study, I. novaecaledoniensis differs substantially from other congeners in possessing a small buccal capsule, by which it shows affinities to species of the dracunculoid family Skrjabillanidae Shigin et Shigina, 1958, parasites of freshwater fishes [25]. It should be noted that skrjabillanids are similar to species of Ichthyofilaria also in having the glandular oesophagus provided with a posterior appendix [25] and the males of both these nematode groups possess a copulatory plate instead of spicules [7,23,25,55,56]. On the other hand, in contrast to I. novaecaledoniensis and other congeners, the skrjabillanid females are monodelphic (vs. didelphic), possessing the vulva situated in the oesophageal region (vs. in the posterior half of body) and the female tail tip of most species bears three projections. Moreover, all skrjabillanids are parasites of freshwater fishes (see above). Consequently, I. novaecaledoniensis is not a species belonging to the Skrjabillanidae.
Besides the presence of a buccal capsule (see above), the female of I. novaecaledoniensis also differs from congeners in the number and arrangement of cephalic papillae. According to Timi et al. [58], there are eight papillae in the outer circle arranged in four submedian pairs and two (but probably four [26]) submedian single papillae in the inner circle in I. argentinensis females (vs. four submedian papillae in the outer circle and four submedian papillae in the inner circle in I. novaecaledoniensis). Four elevated submedian pairs of papillae were reported for females of I. japonica [47], whereas four poorly defined submedian papillae were found in females of I. bergensis and I. canadensis [4,20]; the cephalic papillae of I. dasycotti have not yet been studied.
Considering other dracunculoids, based on the number and arrangement of cephalic papillae in the female, I. novaecaledoniensis resembles only Lucionema balatonense Moravec, Molnár et Székely, 1998 (Lucionematidae), a parasite of the swimbladder wall of the freshwater percid Sander lucioperca (Linnaeus) in Europe and Lockenloia sanguinis Adamson et Caira, 1991 (genus incertae sedis), an inadequately described parasite from the heart of the nurse shark Ginglymostoma cirratum (Bonnaterre) in the western North Atlantic Ocean [1,46]; however, both of these species differ from I. novaecaledoniensis in the structure of the oesophagus and for L. balatonense also in the absence of the buccal capsule and in that its female genital tract is monodelphic (vs. didelphic).
Therefore, we consider it necessary to erect a new genus, Ichthyofilaroides n. gen., to accommodate I. novaecaledoniensis. This new genus is differentiated from Ichthyofilaria mainly by the presence of the buccal capsule and the number and arrangement of the cephalic papillae. Since the buccal capsule has not yet been described in any species of the Guyanemidae, it may be necessary to create a new family for Ichthyofilaroides n. gen. in the future. Nevertheless, since the male of its type species remains unknown, we provisionally assign Ichthyofilaroides to the family Guyanemidae, subfamily Travassosneminae.
It should be noted that, even though the molecular phylogeny of some fish dracunculoids has been studied (e.g. [10,23,50,57,60]), no such studies have so far been performed on representatives of the Guyanemidae.
Site of infection: Unknown with precision, parasites obtained from wash of organs in abdominal cavity.

Description
Male fourth-stage larva (one specimen): Body of specimen undergoing last moult 3.17 mm long, maximum width 30, slightly narrowed posterior to cephalic end (Fig. 3A). Width of cephalic end 24, of narrowed cephalic region 21 and that of caudal end 21. Entire oesophagus 186 long, slightly inflated at anterior end, 15 in maximum width. Nerve ring and cell nucleus of oesophageal gland 57 and 132, respectively, from anterior extremity. Spicules equal, 189 long, representing 6% of body length (Fig. 3B).

Remarks
Ascarophis adioryx has already been reported from two species of holocentrid fish hosts off New Caledonia, Sargocentron spiniferum and Neoniphon sammara (Forsskål) and, on the basis of available specimens, the species was redescribed in detail based on LM and SEM [34]. The present material contained only a single specimen (gravid female); therefore, we have refrained from describing it.
Originally this nematode species was described from S. spiniferum and S. rubrum (Forsskål) in the Philippine Sea off the Palau Islands (Republic of Palau), Oceania [21] and later it was recorded under the synonym Ascarophis holocentri Parukhin, 1984 from S. spiniferum and S. punctatissimum (Cuvier) in the Red Sea [51]. Consequently, A. adioryx seems to be a specific parasite of holocentrid fishes in the Indo-Pacific region.

Remarks
The present specimens are morphologically similar to A. nasonis Machida, 1981, described from the same two hosts species, N. unicornis (now considered to be probably a synonym of N. minor [11]) (type host) and N. lituratus, from off southern Japan (Okinawa Prefecture) and Palau Islands [21] and there is no doubt that they belong to this species. Based on LM, Machida [21] gave a relatively good description of this species, but he could not observe some morphological details visible only with the use of SEM. The present SEM examination of this species revealed, for the first time, the presence of ventral precloacal cuticular ridges in the male, the presence and location of phasmids in the female and showed the exact structure of the nematode's cephalic end. This is the second finding of A. nasonis since its original description [21] and the first record of this nematode from fishes in New Caledonian waters. Previously two other congeneric species were reported from New Caledonia, Ascarophis adioryx from holocentrids (see above) and A. richeri Moravec et Justine, 2007 from the scorpaeniform fish Hoplichthys citrinus [30]. Whereas A. nasonis can be easily distinguished from the otherwise similar A. adioryx by the longer left spicule (810-1100 lm vs. 516-610 lm) and the mouth structure (mouth without dorsal and ventral median projections and pseudolabia without conspicuous inner dorsoventral extensions), both A. adioryx and A. nasonis differ from A. richeri mainly in possessing nonfilamented eggs (vs. eggs with long filaments on either pole) and some other features such as the mouth structure, shape of deirids and the structure of ventral precloacal ridges in the male.
Based on the structure of the mouth, each deirid with two posteriorly oriented arms separated one from another by the body cuticle, an unusually long glandular oesophagus, a fairly long female tail, the obtuse distal tip of the left spicule and the shape of eggs, A. nasonis is similar to the recently described A. scatophagi Moravec, Yooyen et Sanprick, 2018, a parasite of Scatophagus argus (Linnaeus) (Scatophagidae, Perciformes) in the Gulf of Thailand [49]. However, in contrast to A. nasonis, A. scatophagi possesses a distinctly shorter left spicule (555-642 lm vs. 846-1100 lm) and more numerous (about 10 vs. about 4) longitudinal precloacal cuticular ridges; moreover, the hosts of these two nematode species belong to different fish families (Acanthuridae vs. Scatophagidae).
In contrast to the majority of congeneric species, the fully developed (larvated) eggs of A. nasonis were described and illustrated to be conspicuously elongate [21]; similar, markedly elongate-oval eggs were also described for A. longiovata Moravec et Klimpel, 2009 and A. scatophagi [44,49]. Nevertheless, although the fully developed eggs of the present specimens of A. nasonis from N. lituratus are typical in shape of this species (i.e. conspicuously elongate) (Fig. 4J), those of A. nasonis from N. unicornis are relatively wider with respect to their length and some of them appear to bear a small, poorly developed swelling on one pole (Fig. 4K). Since the gravid A. nasonis females from N. unicornis were smaller and less developed (with smaller numbers of eggs in uteri) as compared with those from N. lituratus, it may well be that the egg shape is influenced by the state of the female development.
Ascarophis nasonis has not been recorded previously from New Caledonian waters. Prevalence, intensity and details of fish: 1 fish infected/11 fish examined; 1 nematode. The infected fish, JNC2166, was 595 mm in total length and 3800 g in weight.

Remarks
Since the morphology and measurements of the only available female specimen, including the fine structure of the mouth and the shape of deirids, are much the same as those of Ascarophisnema tridentatum (except for a slightly longer body length) described from the congeneric host Gymnocranius euanus (Günther) from off New Caledonia [35], this specimen is considered to belong to this species. The present finding of A. tridentatum in G. grandoculis represents a new host record.
Yamaguti [61] described Rhabdochona gymnocranii Yamaguti, 1935 from two gravid females collected in the stomach of Gymnocranius griseus (Temminck et Schlegel) from the Inland Sea, Japan and later the species was transferred to Ascarophis van Beneden, 1871 [6]. The species has not been recorded since its original description. Yamaguti's type specimens were re-examined and illustrated by Moravec [24] and Ko [19], but because these were mounted as permanent slides and some taxonomically important features, in particular detailed cephalic structures, could not be studied on them, this species has been considered to be a species inquirenda [35]. Taking into account that Ascarophis sp. of Mamaev, 1970, described from G. griseus of the Gulf of Tonkin near Vietnam [22] which is probably identical with A. gymnocranii [35], is similar to Ascarophisnema tridentatum, the species Ascarophis gymnocranii was provisionally transferred to Ascarophisnema Moravec et Justine, 2010 [35]. It may well be that future studies on A. gymnocranii show its identity with A. tridentatum.
Acknowledgements. We wish to thank all colleagues and students who have participated in the parasitological survey over the years; detailed lists were provided in previous papers [15][16][17]. The deepsea cruise TERRASSES was led by Sarah Samadi. Thanks are also due to the Laboratory of Electron Microscopy, Institute of Parasitology, Biology Centre CAS, institution supported by the MEYS CR (LM2015062 Czech-BioImaging) for their support with obtaining the scientific data presented in this paper, and to Blanka Škoríková of the same Institute for help with the illustrations. This study was partly funded by the institutional support of the Institute of Parasitology, BC AS CR (585110/9500).

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http:// publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jérôme Depaquit.