Three new species of Cucullanus (Nematoda: Cucullanidae) from marine fishes off New Caledonia, with a key to species of Cucullanus from Anguilliformes

Based on light and scanning electron microscopical studies of nematode specimens from the digestive tract of some rarely collected anguilliform and perciform fishes off New Caledonia, three new species of Cucullanus Müller, 1777 (Cucullanidae) are described: C. austropacificus n. sp. from the longfin African conger Conger cinereus (Congridae), C. gymnothoracis n. sp. from the lipspot moray Gymnothorax chilospilus (Muraenidae), and C. incognitus n. sp. from the seabream Dentex fourmanoiri (Sparidae). Cucullanus austropacificus n. sp. is characterized by the presence of cervical alae, ventral sucker, alate spicules 1.30–1.65 mm long, conspicuous outgrowths of the anterior and posterior cloacal lips and by elongate-oval eggs measuring 89–108 × 48–57 μm; C. gymnothoracis n. sp. is similar to the foregoing species, but differs from it in the absence of cervical alae and the posterior cloacal outgrowth, in the shape and size of the anterior cloacal outgrowth and somewhat shorter spicules 1.12 mm long; C. incognitus n. sp. (based on female morphology) differs from other congeneric species parasitic in the Sparidae mainly in possessing cervical alae, the postequatorial vulva, phasmids situated at the mid-length of the tail and in the size of the eggs (75–84 × 45–66 μm). A key to species of Cucullanus parasitizing anguilliform fishes is provided.


Materials and methods
Fish were caught off New Caledonia by various, and sometimes unusual, means. The seabream Dentex fourmanoiri was caught by line; the conger Conger cinereus was taken in a cage baited for the collection of Nautilus; and the moray Gymnothorax chilospilus was obtained from a New Caledonian sea krait, Laticauda saintgironsi Cogger & Heatwole, collected on a small islet, Ilôt Amédée, off Nouméa, New Caledonia. As this host is an emblematic protected species, an indirect sampling method without any effect on survival was used [5]: a gentle massage of the sea krait abdomen provided the stomach content by regurgitation, and the regurgitated contents included the moray eel. Parasites were obtained by a wash method [14]. 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 examination (SEM) 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].
Cucullanus austropacificus n. sp. differs from all of the above-mentioned species, except for C. truttae, in the presence of cervical alae, but also in some other morphological features. By the structure of the cloacal region, the new species is most similar to C. pedroi parasitizing congeneric fish host in the western Atlantic Ocean, but differs from it in the shape (more elongate in C. pedroi) of the oesophastome, its anterior cloacal outgrowth is smaller than the posterior outgrowth (vs. anterior outgrowth larger than the posterior one), the lower posterior part of the posterior cloacal lip is without denticulations (vs. denticulations present) and the sixth pair of subventral papillae is situated posterior to the cloaca (vs. at the level of the cloaca) (see Figs. 60-62 in Vieira et al. [44]). The distinction of C. austropacificus n. sp. from other congeners parasitizing anguilliform fishes is more apparent from the key at the end of the Discussion in this article.
Deposition of type specimen: Helminthological Collection, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Č eské Budějovice, Czech Republic (male holotype mounted on SEM stub, Cat. No. N-1168).
Etymology: The specific name of this nematode relates to the genitive form of the generic name of the host.   Female: Not known.

Remarks
The morphology and measurements of this nematode specimen, as well as the fact that it was collected from the congeneric fish host in the nearby region, show its similarity to C. australiensis, the species originally described by Baylis [2] from Gymnothorax cf. pictus off Australia. Later Morand & Rigby [18] established C. faliexae from G. javanicus in French Polynesia, but it was subsequently synonymized with C. australiensis [30]. Cucullanus australiensis has not yet been studied by SEM, so its detailed morphology remains unknown. Nevertheless, the present specimen differs markedly from C. australiensis in the considerably more posterior situation of deirids and the excretory pore and, therefore, this is considered to represent a separate species. Comparison of C. gymnothoracis n. sp. with other congeneric species is apparent from the key presented at the end of the Discussion in this article.     Male: Not known.

Remarks
Even though males of this new species are not known, C. incognitus n. sp. can be distinguished from the great majority of Cucullanus spp. by the presence of lateral cervical alae. Of the many species of Cucullanus, the presence of cervical alae, as found in C. incognitus, has hitherto been described only in C. truttae, a parasite mainly of freshwater salmonids (Salmonidae) in the Holarctic [21,28], and in two recently established species parasitizing marine fishes in New Caledonian waters, i.e., C. epinepheli Moravec & Justine, 2017 parasitic in Epinephelus chlorostigma (Valenciennes) (Serranidae, Perciformes) and C. austropacificus n. sp. from Conger cinereus reported in the present paper. Rasheed [38] mentioned the presence of asymmetrical ''cuticular expansions in the form of cephalic alae'' in C. theraponi Rasheed, 1968 from ''Therapon'' (= Terapon?) sp. (Terapontidae, Perciformes) and Hilsa sp. (Clupeidae, Clupeiformes) from off Pakistan, but these formations are different from cervical alae.
Cucullanus incognitus n. sp. differs from C. epinepheli in that its posterior portion of the oesophagus is narrower (vs. markedly wider) than the anterior oesophastome, its deirids are situated more anteriorly in relation to the length of the oesophagus (at 68% vs. 77-86% of oesophagus length) and the hosts belong to different fish families (Sparidae vs. Serranidae). In contrast to C. austropacificus, the gravid females of the new species are much smaller (body length approximately 13-15 mm vs. 26-35 mm), their oesophastome is more elongate (approximately twice as long as wide vs. approximately as long as wide) and their eggs are smaller (75-84 · 45-66 lm vs. 84-108 · 48-57 lm) and of a different shape (oval vs. elongate-oval); hosts of these two species belong to different fish orders (Perciformes vs. Anguilliformes). Regarding C. truttae, it can be easily distinguished from C. incognitus by the conspicuously asymmetrical cephalic end and by the excretory pore located at the mid-distance between the nerve ring and the oesophago-intestinal junction (vs. excretory pore posterior to the oesophageal end).
To date, the only nominal species of Cucullanus previously described from seabreams (Sparidae) are C. chrysophrydis Gendre, 1928, parasitizing Pagellus bogaraveo (Brünnich) and Sparus aurata Linnaeus off the Atlantic coast of Africa [7,13,43], and C. protrudens Pereira, Vieira & Luque in Vieira et al., 2015, a parasite of Pagrus pagrus (Linnaeus) from off the Atlantic coast of Brazil [44]. Both these species differ from C. incognitus n. sp. in the absence (vs. presence) of cervical alae and in the location of phasmids in the second half of the tail (vs. at mid-length of tail). In addition, the vulva of C. protrudens is preequatorial, at 41%-43% of the body length (vs. postequatorial in the new species, at 60%-61% of body length). Moreover, all these three nematode species parasitize hosts belonging to different genera (Pagellus Valenciennes, Pagrus Cuvier and Sparus Linnaeus vs Dentex Cuvier) and they occur in geographically very distant regions (C. chrysophrydis and C. protrudens in the Atlantic Ocean vs C. incognitus n. sp. in the Pacific Ocean).
An unidentified species of Cucullanus, Cucullanus sp. from Pagrus sp., was reported by Vassiliadès [43] in the list of helminth parasites of marine fishes off the coast of Senegal (Atlantic Ocean). Cucullanus sp. was also reported from Pagrus auratus (Forster) in the Pacific Ocean, New Zealand [39]. However, in this case, specimens of another cucullanid genus Dichelyne Jägerskiöld, 1902 were probably misidentified as Cucullanus, as indicated by the small body measurements and accompanying illustrations (the intestinal caecum in Dichelyne spp. is sometimes difficult to observe and was probably overlooked by the authors). This is also supported by the fact that Cucullanellus (= Dichelyne) cnidoglanis Johnston & Mawson, 1945 was reported from the same host species (P. auratus) in the same region (off New Zealand) [6]. Three species of the Sparidae, Acanthopagrus schlegelii (Bleeker), Dentex (reported as Evynnis Jordan & Thompson) tumifrons (Temminck & Schlegel) and Pagrus major (Temminck et Schlegel), were reported as hosts of Dichelyne jialaris Luo, Guo, Fang & Huang, 2004 from off China and Japan [16,29].
The authors are aware of the fact that the description of C. incognitus n. sp. is based solely on female morphology, a procedure that cannot generally be recommended; however, in this case, the new species appears to be well established and, therefore, we consider it useful to give the species a name rather than to report it as Cucullanus sp. and to wait years until conspecific males are available; the host is extremely rarely collected.
It should be noted that the only host specimen (D. fourmanoiri) examined harboured, in addition to C. incognitus n. sp., two specimens of the cystidicolid nematode Rasheedia heptacanthi Moravec & Justine, 2018 in the digestive tract [25].

Discussion
As mentioned above, the morphology of the numerous species of Cucullanus is rather uniform. Therefore, the separation of similar species based solely on morphological features studied by LM may be problematic, especially in the situation when some Cucullanus spp. have been insufficiently described. Nevertheless, some papers published during last two decades F. Moravec and J.-L. Justine: Parasite 2018, 25, 51 (e.g. [15, 17, 22-24, 27, 30, 32-34, 41, 42, 44]) have shown the importance of the use of SEM for the taxonomy of these nematodes, because some features are difficult to observe or are not visible at all under the LM. This concerns, for example, the exact number and distribution of caudal papillae in the male or the situation of deirids and the excretory pore. One such feature is the presence of narrow lateral cervical alae, observable in dorsoventral view, which can be easily overlooked when using LM, but their presence can be confirmed by SEM. The presence/absence of cervical alae appears to be an important specific taxonomic feature in Cucullanus; as stated above, of many described species of Cucullanus, cervical alae have hitherto been reported only in C. austropacificus n. sp., C. epinepheli, C. incognitus n. sp. and C. truttae. According to experimental observations [19], cervical alae of third-and fourth-stage larvae of C. truttae are much wider than those of conspecific adults.
As revealed by SEM, taxonomically very important features are found in the structures of the cloacal region in the male. It seems that many species of Cucullanus possess a small, rounded median precloacal elevation, sometimes reported as the median precloacal papilla or the median precloacal organ; in fact, such an elevation usually bears a single minute papilla (e.g. in C. gymnothoracis n. sp., see Fig. 6D) or, less often, two minute papillae (C. epinepheli, C. genypteri Sardella, Navone & Timi, 1997) are visible on its surface [44], present paper. However, there are species of Cucullanus (e.g. C. bulbosus), in which the median precloacal elevation is lacking [22].
Consequently, when studying cucullanid species, as well as other nematodes, it is highly desirable to examine them by both LM and SEM. Of course, the use of molecular methods, if possible, is also important.
Key to species of Cucullanus parasitic in anguilliform fishes (Anguilliformes): Median outgrowth adherent to inner rim of anterior cloacal lip distinctly smaller than median precloacal papilla-like formation. Posterior cloacal lip without conspicuously elevated outgrowth. Spicules 668-1020 lm, spicular alae not conspicuously broad. Body length of male approximately 6-10 mm, that of gravid female 9-14 mm.