New records of anisakid nematodes from marine fishes off New Caledonia, with descriptions of five new species of Raphidascaris (Ichthyascaris) (Nematoda, Anisakidae)

Recent examinations of anisakid nematodes (Anisakidae) from marine fishes off New Caledonia, collected in the years 2003–2008, revealed the presence of the following five new species of Raphidascaris Railliet et Henry, 1915, all belonging to the subgenus Ichthyascaris Wu, 1949: Raphidascaris (Ichthyascaris) spinicauda n. sp. from the redbelly yellowtail fusilier Caesio cuning (Caesionidae, Perciformes); Raphidascaris (Ichthyascaris) fasciati n. sp. from the blacktip grouper Epinephelus fasciatus (Serranidae, Perciformes); Raphidascaris (Ichthyascaris) nudicauda n. sp. from the brushtooth lizardfish Saurida undosquamis (Synodontidae, Aulopiformes); Raphidascaris (Ichthyascaris) euani n. sp. from the Japanese large-eye bream Gymnocranius euanus (Lethrinidae, Perciformes); and Raphidascaris (Ichthyascaris) elopsis n. sp. from the Hawaiian ladyfish Elops hawaiensis (Elopidae, Elopiformes). An additional two congeneric species, R. (I.) etelidis Moravec et Justine, 2012 and R. (I.) sillagoides (Bruce, 1990) were found in the deep-water red snapper Etelis carbunculus (new host record) and the deepwater longtail red snapper Etelis coruscans (both Lutjanidae, Perciformes), and the silver sillago Sillago sihama (Sillaginidae, Perciformes) (new host and geographical records), respectively. Two unidentified congeneric species, Raphidascaris (Ichthyascaris) sp. 1 from the trumpet emperor Lethrinus miniatus (Lethrinidae, Perciformes) and Raphidascaris (Ichthyascaris) sp. 2 from the white-spotted puffer Arothron hispidus (Tetraodontidae, Tetraodontiformes) were recorded. Moreover, two species of Hysterothylacium Ward et Magath, 1917, H. alatum Moravec et Justine, 2015 and H. epinepheli (Yamaguti, 1941), were found in the leopard coralgrouper Plectropomus leopardus (type host) and the highfin grouper Epinephelus maculatus (new host) (both Serranidae, Perciformes), respectively. This is the second finding of H. epinepheli since its original description in Japan 79 years ago. Most species are described based on light and electron microscopical studies.

Recent examinations of adult anisakid nematodes collected by J.-L. Justine and his students in marine fishes from off New Caledonia in the years 2003-2008 revealed the presence of five previously unknown and one known species of Raphidascaris Railliet et Henry, 1915 (subgenus Ichthyascaris Wu, 1949), the latter representing new host and geographical records, and two known species of Hysterothylacium Ward et Magath, 1917, the finding of one of which also represents new host and geographical records. Results of this study are presented herein.

Materials and methods
Fish were caught off New Caledonia by various means; those obtained from the fishmarket in Nouméa were very fresh and thus were probably fished in the near vicinity. The nematodes were generally collected with the "wash" method and were fixed in hot 4% formalin or 70% ethanol [13]. For light microscopical (LM) examination, 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 classification system of the Ascaridoidea adopted follows Keys to the Nematode Parasites of Vertebrates [1,7]. The fish nomenclature follows FishBase [6]. Oesophagus short; posterior half markedly broad in female (Fig. 1A). Ventriculus transversely oval; ventricular appendix relatively short (Fig. 1A). Excretory pore well posterior to level of nerve ring (Fig. 1A). Tail of both sexes conical.

Results
Male Female ( [14,19]. Li et al. [14] also listed R. lutiani Olsen, 1952 (misspelled as lutjani) in this subgenus, but the main morphological feature of Ichthyascaris (i.e., anteriorly united lateral alae) [22] is not apparent from the original description of this North American species [24], so that it is not included in this subgenus.
Sheenko [28] designated R. lophii and R. trichiuri as the junior synonyms of R. chirocentri, but this conclusion was based solely on available inadequate species descriptions. Therefore, both these species are, for the time being, dealt with as valid in the present paper. The two males and one female of Raphidascaris (Ichthyascaris), collected from the intestine of Etelis carbunculus Cuvier (reported as E. marshi) (Lutjanidae) off the Philippines, were identified and briefly described by Sheenko [28] as Ichthyascaris [= R. (I.)] chirocentri, but this identification seems to be questionable.
The new species, R. (I.) spinicauda n. sp., differs distinctly from R. fisheri and R. trichiuri in the presence of numerous small cuticular spines on the female tail tip; from the former species also by the absence of a small bulge posterior to the anterolateral sockets on the lateral margins of the lips [5,16]. From R. lophii and R. longispicula it differs in much shorter spicules (309 lm vs. 540-690 lm and 1.13-1.32 mm, respectively) [14,30], from R. vicentei it can be differentiated by the male tail tip with spines (vs. aspinose), slightly longer spicules (309 lm vs. 125-300 lm), less numerous pairs of postanal papillae (8 pairs vs. 10-11 pairs) and by less elongate lips with protruding inner lobes (vs. more elongate lips without markedly protruding inner lobes), whereas from R. chirocentri by less numerous pairs of all caudal papillae and those of postanal papillae (43 and 8 vs. 63 and 13) [29,31].
In contrast to the new species, the male tail tip of R. nemipteri is smooth (vs. spinose) and the postanal papillae of the third pair from the posterior extremity are doubled (vs. single) [17]. The male tail tip of R. arii, R. etelidis, R. gymnocraniae, and R. sillagoides is spinose as in the new species, but the postanal papillae of the third pair from the posterior extremity in R. gymnocraniae and R. sillagoides are single (vs. doubled), and they have fewer pairs of all caudal papillae (33-38 and 31-37, respectively, vs. 43) [5]. The postanal papillae of the third pair from the posterior extremity in R. arii and R. etelidis are doubled (vs. single); moreover, the former species differs in fewer pairs of preanal papillae (21-30 vs. 43) and the latter one in longer spicules (345-474 lm vs. 309 lm), and more pairs of preanal (44-49 vs. 35) and postanal (12-13 vs. 8) papillae [19,33]. Raphidascaris spinicauda n. sp. is the first species of the subgenus Ichthyascaris reported from a fish host belonging to the family Caesionidae.  Prevalence, intensity and details about fish: 1 fish infected/ 61 fish examined [12]; 92 nematodes. The infected fish, JNC2280, was 280 mm in fork length and 326 g in weight.
Etymology: The specific name of this nematode relates to the genitive form of the species name of the type host.

Description
General: Medium-sized nematodes with transversely striated cuticle (Figs. 4C, 4D and 5C). Lips approximately equal in size, without lateral membranous flanges; pulp with 2 moderately developed anterior lobes, each with terminal pocket-like depression. Dorsal lip bears 2 subdorsal double papillae (Fig. 4A); each ventrolateral lip with 1 double subventral papilla, 1 small single papilla and amphid situated laterally. Interlabia absent. Narrow lateral alae extend along whole body length, united anteriorly close to ventrolateral lips on 1 side of body (Fig. 3A). Oesophagus short; posterior half markedly broad (Fig. 3A). Ventriculus transversely oval; ventricular appendix relatively short  This new species is characterized by the absence of cuticular outgrowths (spines or protuberances) on the male tail tip (Figs. 3E and 5C). Of the 11 species of Raphidascaris (Ichthyascaris) (see above), only the following five have the male tail tip smooth, without minute cuticular spines or protuberances: R. chirocentri, R. fisheri, R. nemipteri, R. trichiuri, and R. vicentei. However, in contrast to the new species, the female tail tip of R. fisheri and R. trichiuri is smooth, without cuticular spines (vs. female tail tip with numerous small spines); moreover, R. fischeri has lateral margins of the lips with a small bulge posterior to the anterolateral sockets [5] (vs. such structures not present). The remaining three species, R. chirocentri, R. nemipteri, and R. vicentei, have shorter spicules (315 lm, 225-399 lm and 125-300 lm, respectively, vs. 420-543 lm) and also differ in some other features, such as the numbers of genital papillae and body measurements. The type hosts of these five species belong to other fish families than that of the new species (Chirocentridae, Muraenesocidae, Nemipteridae, and Platycephalidae vs Serranidae).
Epinephelus fasciatus has been thoroughly examined for parasites in New Caledonia with 21 host-parasite combinations reported in 2010 [10], which were subsequently complemented by additional records of nematodes [20], trypanorhynch cestodes [2] and copepods [8]. The description of the present new species exemplifies again the high biodiversity of parasites in coral-reef fish.
Raphidascaris (Ichthyascaris) nudicauda n. sp. Prevalence, intensity and details about fish: 1 fish infected/6 fish examined the same day; 1 nematode. The infected fish, JNC2591, was 182 mm in length and 52 g in weight.
Etymology: The specific name nudicauda is the Latin noun in apposition, composed of two words, nudus (= bare, naked) and cauda (= tail), and relates to the characteristic feature of this species, i.e. the absence of cuticular spines on the male tail tip.
Deposition of type specimen: Holotype mounted on SEM stub in the Helminthological Collection, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (Cat. No. N-1216). Female: Not known.

Remarks
This new species has no cuticular spines or protuberances on the male tail, as visible in Figure 11B (this being more apparent when further enlarged). Of the 12 species of Raphidascaris (Ichthyascaris) (see above), only R. chirocentri, R. fasciati n. sp., R. fisheri, R. nemipteri, R. trichiuri, and R. vicentei have the male tail tip smooth, without minute cuticular spines or protuberances. However, in contrast to R. nudicauda n. sp., R. chirocentri has longer spicules (315 lm vs. 201 lm), more numerous pairs of all caudal papillae (63 vs. 36) and those of   [29]. Raphidascaris nudicauda n. sp. is the first representative of the subgenus Ichthyascaris reported from a fish belonging to the aulopiform family Synodontidae. Larval anisakids were reported from this fish [25,27].
Etymology: The specific name of this nematode relates to the genitive form of the species name of the type host.
The only species of the subgenus Ichthyascaris reported from hosts belonging to the perciform family Lethrinidae is R. gymnocraniae described from Gymnocranius audleyi Ogilby (reported as G. bitorquatus Cockerell) (type host) and Lethrinus miniatus (Forster) (reported as L. chrysostomus Richardson) recorded from off the western Pacific coast of Australia (Queensland) [5]. Despite the fact that the present New Caledonian specimens were collected from the fish of the same genus (Gymnocranius Klunzinger) as the type host of R. gymnocraniae and from the nearby region, their morphology is very different (see above) and, consequently, they are considered to represent a new species.
Gymnocranius euanus has been thoroughly examined for parasites in New Caledonia, with 23 host-parasite combinations reported in 2010 [11]. Additional records since include digeneans [4], copepods [8] and larval anisakids [25]. The description of the present new species exemplifies again the high biodiversity of parasites in coral fish.   Prevalence, intensity and details about fish: 1 fish infected/ 1fish examined; c. 30 nematodes. The fish, JNC848, was 980 mm in fork length and 7000 g in weight.
Etymology: The specific name of this nematode relates to the genitive form of the generic name of the type host.

Remarks
Only seven species of Raphidascaris (Ichthyascaris), R. arii, R. etelidis, R. euani n. sp., R. gymnocraniae, R. longispicula, R. sillagoides, and R. spinicauda n. sp., have the tail tip of both males and females covered by many small cuticular spines or protuberances, as in the new species. Of them, R. longispicula differs by considerably longer spicules (1.13-1.32 mm), whereas the spicule lengths of the remaining six species are similar to those of R. elopsis n. sp. However, in contrast to the new species, the total number of pairs of caudal papillae is much higher in R. etelidis and R. euani (60-65 and 60, respectively, vs. 38-43); moreover, these two species also have more numerous pairs of postanal papillae (12-13 and 11, respectively, vs. 8). Whereas postanal papillae of the third pair from the posterior extremity are single in the new species as in R. gymnocraniae, R. sillagoides and R. spinicauda, these are double in R. arii and R. etelidis; postanal papillae of the same pair are single in R. euani, but those of the second pair from the posterior extremity are triple (vs. single).
Consequently, R. elopsis n. sp. cannot be differentiated from R. gymnocraniae, R. sillagoides and R. spinicauda based on the above-mentioned morphological features. However, the pairs of preanal papillae of R. gymnocraniae and R. sillagoides are less numerous (24-28 and 22-26, respectively, vs. 29-35), whereas those of postanal papillae may be more numerous (7-9 and 8-10, respectively, vs. 8); the vulva of gravid R. sillagoides females is located more posteriorly as compared to the new species (at 23-29% vs. 14-21% of the body length from the anterior extremity). The number of preanal papillae in R. spinicauda is identical to that in the new species (35 vs. 29-35), as well as that of postanal papillae (8 vs. 8), but both species differ from each other in the shape and structure of the female tail tip. Whereas the female tail tip of R. spinicauda is conical, sharply pointed, all covered with cuticular spines (Figs. 1C and 2D), that of R. elopsis n. sp. is truncated, with several outgrowths located on its top (Fig. 14C). Moreover, the hosts of these two species belong to different fish families and orders (Caesionidae, Perciformes vs Elopidae, Elopiformes).

Remarks
This species has already been described in detail by Moravec and Justine [19] from lutjanid fishes E. coruscans (type host) and Pristipomoides filamentosus (Valenciennes) off New Caledonia. Whereas the present material from E. carbunculus contained both males and females, that from E. coruscans consisted only of a young female and one third-stage larva. The finding of this nematode species in E. carbunculus represents a new host record. The general morphology of the only available specimen is in agreement with the description of R. (I.) sillagoides, the only species of the subgenus Ichthyascaris parasitizing fishes of the family Sillagonidae [5]. The situation of the vulva in the present specimen (at 18% of the body length) as compared to that reported for R. sillagoides (23-29%) is apparently due to the fact that the New Caledonian specimen is represented by a small-sized, nongravid female, whereas the gravid females of R. sillagoides may be up to about 30 mm long. Taking into account that the present nematode was found in the congeneric host in the nearby region, it is considered to belong to R. sillagoides.

Raphidascaris
Raphidascaris (I.) sillagoides was described from Sillago maculata Quoy et Gaimard from off the western Pacific coast of Australia [5] and has not been recorded since. Accordingly, the present finding of this species from S. sihama in New Caledonia represents new host and geographical records. Prevalence, intensity and details about fish: 1 fish infected/ 27 fish examined (Justine et al. [11]); 20 nematodes. The infected fish, JNC2824, was 430 mm in fork length and 1600 g in weight.

Remarks
Only a single juvenile male and numerous third-and fourthstage larvae were collected from L. miniatus. Since no nominal species of Raphidascaris (Ichthyascaris) has so far been reported from fishes of the family Lethrinidae, it is highly probable that the present specimens belong to a new, undescribed species. In New Caledonia, Raphidascaris (I.) larvae (rarely also poorly preserved adults) were previously recorded from Lethrinus genivittatus Valenciennes, L. miniatus and L. rubrioperculatus Sato [19], which might belong to the same nematode species.

Remarks
One gravid female and two body fragments of small larvae were collected from the digestive tract of A. hispidus. Considering the host specificity of Raphidascaris (Ichthyascaris) species at the level of host fish families and the fact that previously no representatives of this anisakid subgenus were recorded from tetraodontiform fishes, apparently the present specimens belong to an undescribed species. The available female is the longest one among all known species of Ichthyascaris, only the female of R. (I.) trichiuri may nearly reach the same length (up to 37.8 mm), but its tail tip lacks minute cuticular spines. Nevertheless, in having no males, we refrain from establishing a new species for these nematodes. Prevalence, intensity and details about fish: 1 fish infected/ 24 fish examined [10]; 1 nematode. The infected fish specimen, JNC381, was 285 mm in total length and 301 g in weight.

Remarks
The only available specimen (young gravid female) is morphologically identical to those of H. alatum, described from the congeneric host Plectropomus laevis (Lacepède) off New Caledonia [21], and, consequently, it is considered to belong to this species. The present finding of H. alatum in P. leopardus represents a new host record for this nematode species.