New records of cucullanid nematodes from marine fishes off New Caledonia, with descriptions of five new species of Cucullanus (Nematoda, Cucullanidae)

Recent examinations of cucullanid nematodes (Cucullanidae) from marine fishes off New Caledonia, collected in the years 2004–2009, revealed the presence of the following five new species of Cucullanus Müller, 1777, all parasitic in Perciformes: Cucullanus variolae n. sp. from Variola louti (type host) and V. albimarginata (both Serranidae); Cucullanus acutospiculatus n. sp. from Caesio cuning (Caesionidae); Cucullanus diagrammae n. sp. from Diagramma pictum (Haemulidae); Cucullanus parapercidis n. sp. from Parapercis xanthozona (type host) and P. hexophtalma (both Pinguipedidae); and Cucullanus petterae n. sp. from Epinephelus merra (type host) and E. fasciatus (both Serranidae). An additional congeneric species, Cucullanus bioccai Orecchia et Paggi, 1987 was recorded from Mugil cephalus (Mugilidae, Mugiliformes) (first record in the Pacific Ocean) and Cucullanus sp. (only female) was found in Arothron manilensis (Tetraodontidae, Tetraodontiformes). Furthermore, two known cucullanid species, Dichelyne (Cucullanellus) branchiostegi (Yamaguti, 1941) in Branchiostegus wardi (Malacanthidae, Perciformes) (new host and geographical records) and Dichelyne (Cucullanellus) bodiani Moravec et Justine, 2019 in Bodianus busellatus (new host) and B. perditio (both Labridae, Perciformes), were found; Dichelyne (Cucullanellus) sp. (only females) coinfecting the latter host may represent an unknown species. Most species are described based on light and electron microscopical studies. The specimens described by Xu et al., 2017 as Cucullanus bourdini Petter et Le Bel, 1992 from Caesio xanthonota (Caesionidae) in the Taiwan Strait are considered to represent a new species, for which the name Cucullanus sinensis n. sp. is proposed.


Introduction
A total of eight nominal species of cucullanid nematodes (Cucullanidae) has so far been recorded from marine anguilliform, perciform and tetraodontiform fishes off New Caledonia: Three of the newly established species of Cucullanus have been described from the male holotypes collected in the respective type host species, whereas morphologically and biometrically very similar females collected from sympatric congeneric hosts are considered to belong to each of them. The conspecificity of females and males in these cases, taking into account the usually wider host specificity in Cucullanus spp., is almost certain. Nevertheless, the authors are aware of the fact that it cannot be entirely ruled out, although this is highly improbable, that subsequent studies might show that the females in question do not belong to the newly described species. However, according to the International Code of Zoological Nomenclature, each species is objectively determined by its holotype; the principle of type series helps to solve the situation when specimens of more than one species were included under the same species name. The present authors consider it more reasonable and useful to assign the abovementioned nematode females to the newly described species than to report them only as Cucullanus spp.

Materials and methods
Fish were caught off New Caledonia by various means; those obtained from the fishmarket in Nouméa were very fresh and were thus probably fished in the near vicinity, the same day. 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-pointdried 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 [3].

Remarks
Because of taxonomic problems concerning numerous species of Cucullanus due to their rather uniform morphology and often inadequate descriptions, a detailed comparison among all of them is impossible [18]. Therefore, these parasites are mostly dealt with according to their host groups or the geographical region of their distribution, e.g. [14,17,22]. At present, only two valid nominal species of Cucullanus parasitizing the Serranidae are known [14]: C. mycteropercae Mejía-Madrid et Guillén-Hernández, 2011 from Mycteroperca bonaci Poey from off the coast of Yucatán, Mexico and C. epinepheli from Epinephelus chlorostigma (Valenciennes) off New Caledonia [7,14]. However, both C. mycteropercae and C. epinepheli possess a distinct posterior outgrowth on the elevated anterior cloacal lip, which is absent in the new species, and the precloacal median elevation of C. mycteropercae and C. epinepheli has one or two small papillae, respectively (vs. no papillae present on the precloacal median elevation in the new species).
Moreover, in contrast to C. variolae n. sp., C. epinepheli is characterized by the presence (vs. absence) of narrow lateral cervical alae, the posterior part of the oesophagus broader (vs. narrower) than the pseudobuccal capsule, longer spicules (748-789 lm vs. 510 lm) representing 9-10% (vs. 5.5%) of the body length and the excretory pore located somewhat posterior (vs. anterior) to the level of the posterior end of the oesophagus. Cucullanus mycteropercae also differs from the new species in the presence (vs. absence) of postdeirids, the more posterior location of deirids relative to the level of the oesophago-intestinal junction and in the excretory pore situated distinctly posterior (vs. anterior) to the oesophagus end. In addition, C. variolae n. sp. differs from both these species in the genus of fish hosts (Variola Swainson vs. Epinephelus Bloch or Mycteroperca Gill) and from C. mycteropercae also in the geographical region (South Pacific Ocean vs. North Atlantic Ocean).

Remarks
To date, the only species of Cucullanus reported from a fish host belonging to the perciform family Caesionidae is C. bourdini. This species was originally described from three species of lutjanid fishes off New Caledonia [23] and, subsequently, C. bourdini was reported from three host species belonging to the Lutjanidae, Holocentridae and Balistidae from off French Polynesia [8]. Recently, C. bourdini was reported from the tetraodontid fish Arothron hispidus (Linnaeus) from off Palmyra Atoll, East Indo-Pacific [4]. Nevertheless, Moravec and Justine [13], based on LM and SEM examinations of specimens newly collected from two congeneric lutjanid hosts (Pristipomoides spp., including the type host P. filamentosus (Valenciennes)) in New Caledonia, redescribed C. bourdini  and indicated a certain host specificity of this nematode within the sympatric species of the Lutjanidae. Xu et al. [27], with reference to the descriptions of C. bourdini by Petter and Le Bel [23] and Moravec and Justine [13], identified nematodes collected from Caesio xanthonota Bleeker (Caesionidae, Perciformes) in the Taiwan Strait as C. bourdini, mentioning that Caesionidae is closely related to the Lutjanidae. Nevertheless, the authors found some morphological differences as compared with the previous descriptions of C. bourdini, which they considered to be within intraspecific variability. However, although the general morphology and measurements of their nematodes seem to be similar to those of C. bourdini, the location of the excretory pore and deirids to the oesophago-intestinal junction, considered an important taxonomic feature in Cucullanus spp., is very different (deirids and excretory pore conspicuously far posterior to the oesophagus end versus deirids at level of the oesophagointestinal junction or slightly anterior or posterior to it, and excretory pore short distance posterior to deirids in C. bourdini). Therefore, in our opinion, the specimens from Caesio xanthonota represent a new Cucullanus species, different from C. bourdini, for which the name Cucullanus sinensis n. sp. is proposed. The new name has been registered in ZooBank as urn:lsid:zoobank.org:act:1A049B06-D1B8-4CC1-9BA0-5104E710A37D.
Generally, the morphology of C. acutospiculatus n. sp., for example the shape and structure of the oesophagus, the presence of a ventral sucker and a simple median precloacal elevation, the absence of a posterior outgrowth on the anterior cloacal lip, the number and distribution of caudal papillae or the location of the excretory pore and deirids relative to the oesophago-intestinal junction, is similar to that of C. bourdini; except for the location of deirids and the excretory pore (see above), also to that of C. sinensis n. sp. However, the males of C. acutospiculatus n. sp. are much shorter than those of C. bourdini and C. sinensis There are c. 70 peribuccal denticles in C. acutospiculatus n. sp., but c. 90 denticles in each C. bourdini and C. sinensis. The nerve ring encircles the oesophagus at 39-43% of its length in C. acutospiculatus n. sp., whereas at 32-36% and 35-36% in C. bourdini and C. sinensis, respectively. In addition, C. acutospiculatus n. sp. differs from C. bourdini in the family of the fish host (Caesionidae vs. Lutjanidae) and from C. sinensis in the geographical region (off New Caledonia, South Pacific Ocean vs. Taiwan Strait, western North Pacific Ocean) and the host species (Cae. cuning vs. Cae. xanthonota).   (Fig. 7D). Cloacal region not elevated. Spicules equal, 315 long, representing 5.9% of body length. Gubernaculum 66 long, rod-like in lateral view (Fig. 7D). Caudal papillae 10 pairs: 5 pairs of subventral preanal papillae and 5 pairs of postanal papillae (3 subventral and 2 lateral and dorsolateral); last 2 preanal and 1st postanal pairs of subventrals close to each other; first lateral pair of postanals slightly posterior to level of cloacal aperture; papillae of dorsolateral postanal pair slightly posterior to level of 2nd pair of subventrals (Fig. 7D). Pair of small lateral phasmids somewhat anterior to level of 2nd subventral  pair of postanal papillae (Fig. 7D). Length of tail 286 (Fig. 7D).
To date, C. diagrammae n. sp. seems to be the only nominal species of Cucullanus parasitizing a host of the perciform family Haemulidae. Prevalence, intensity and details about fish: P. xanthozona: 1 fish infected/2 fish examined; 1 nematode; the infected fish (JNC2252) was 165 mm in fork length and 43 g in weight. P. hexophtalma: 1 fish infected/2 fish examined; 1 nematode; the infected fish (JNC2537) was 210 mm in fork length and 96 g in weight.
Deposition of type specimens: Helminthological Collection, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (holotype and allotype mounted on SEM stubs, IPCAS N-1220).
Etymology: The specific name of this nematode relates to the genitive form of the generic name of the hosts. Female (1 gravid specimen from P. hexophtalma, allotype): Length of body 4.62 mm, maximum width 204; width at level of oesophastome 136, at middle of oesophagus 109. Length of entire oesophagus 517, representing 11% of whole body length; length of oesophastome 163, its width 122; minimum width of oesophagus 54; maximum width of posterior part of oesophagus 109 (Fig. 9B). Distance of nerve ring from anterior extremity 204, representing 39% of oesophageal length. Deirids and excretory pore 476 and 666, respectively, from anterior end of body (Fig. 9B). Vulva postequatorial, 2.58 mm from anterior extremity, at 56% of body length; vulval lips not elevated. Vagina directed anteriorly from vulva. Uterus contains many eggs; eggs oval, thin-walled, size 69-72 Â 45-48, with uncleaved contents (Fig. 9D). Tail 153 long, with pointed tip; small lateral phasmids situated approximately at its middle (Figs. 9G, 11C).

Remarks
To date, only two nominal species of Cucullanus have been described from fishes belonging to the perciform family   Prevalence, intensity and details about fish: E. merra: 1 fish infected/18 fish examined [5]; 1 nematode; the infected fish (JNC1649) was 170 mm in fork length and 66 g in weight. E. fasciatus: 1/61 fish examined [5]; 1 nematode; the infected fish (JNC1658) was 180 mm in fork length and 88 g in weight.
Deposition of type specimens: Helminthological Collection, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (holotype and allotype mounted on SEM stubs, IPCAS N-1221).

Remarks
Of the three nominal species of Cucullanus parasitizing serranid fishes, i.e. C. epinepheli, C. mycteropercae and C. variolae n. sp. (see above), only C. epinepheli possesses a large cuticular outgrowth on the anterior cloacal lip and two papillae on the median precloacal elevation as the new species. However, in contrast to C. petterae n. sp., the anterior cloacal outgrowth of C. epinepheli extends posteriorly to cover the cloacal aperture (vs. cloacal outgrowth not covering the cloaca) and there is no elevation on the posterior cloacal lip (vs. posterior cloacal lip bears a conspicuous broad elevation). Moreover, C. epinepheli also differs in the presence (vs. absence) of cervical alae, longer spicules (748-789 lm vs. 501 lm) and the body length of males (7.9-9.0 mm vs.  Prevalence, intensity and details about fish: 1 fish infected/5 fish examined; 2 nematodes; the infected fish (JNC2180) was 465 mm in fork length and 1,730 g in weight.

Remarks
Since conspecific males are absent, the species identification of these nematodes is based solely on female morphology. Both the morphology and measurements of these nematodes are very close to those of C. bioccai, as described from the same host species, M. cephalus, in Italy [20] and are, therefore, considered to belong to this species. Although the deirids of C. bioccai were illustrated as being located at the level of the oesophagus posterior end, these are largely situated anteriorly to the end of the oesophagus according to the original species description [20]. This is the first record of C. bioccai from off New Caledonia and in the region of the Pacific Ocean. The presence of this parasite, originally described in Italy, in New Caledonian waters is not surprising, because its host, M. cephalus, is cosmopolitan in coastal waters of the tropical, subtropical and temperate zones of all seas [3]. It is known that the distribution area of a parasite often coincides with that of its definitive host. Similar cases when the same parasite was recorded from the same host species both in the Atlantic and Pacific regions have recently been reported by Barton et al. [1] and Moravec and Barton [9] for the acanthocephalan Serrasentis sagittifer (Linton, 1889) and the nematode Digitiphilometroides marinus (Moravec et de Buron, 2009), respectively, both parasitizing the marine fish (cobia) Rachycentron canadum (Linnaeus). Also the nematode Piscicapillaria bursata Moravec et Barton, 2019, a parasite of hammerhead sharks (Sphyrna spp.) described from off Australia [10] has recently been recorded from hammerheads off the Atlantic coast of South Carolina, USA [11]. Prevalence, intensity and details about fish: 1 fish infected/2 fish examined; 1 nematode; the infected fish (JNC2422) was 272 mm in fork length and 400 g in weight.

Remarks
Species identification was impossible, because only a single, not well-preserved female specimen was collected from this host. To date, only five nominal species of Cucullanus have been reported from tetraodontiform fishes, of which only C. dodsworthi Barreto, 1922 is known from hosts of the Tetraodontidae from Atlantic waters of Brazil, Mexico and Africa [6], whereas C. bourdini, C. brevicaudatus Pereira, Vieira et Luque, 2014, C. hansoni and C. longipapillatus Olsen, 1952 were reported from fishes of the Balistidae in French Polynesia, from off the Atlantic coast of Brazil, off Hawaii and New Caledonia, and off Hawaii, respectively [8,16,19,21]. However, Moravec and Justine [16] considered the record of C. bourdini, a parasite of perciform fishes, from Balistapus undulatus (Park) (Balistidae) by Morand and Rigby [8] as probably based on misidentification. In view of a certain degree of host specificity of cucullanids, the present Cucullanus sp. from A. manilensis may belong to an undescribed new species.