Philometrids (Nematoda: Philometridae) in carangid and serranid fishes off New Caledonia, including three new species

A recent examination of newly obtained specimens of philometrid nematodes (Philometridae) parasitising carangid and serranid fishes off New Caledonia, South Pacific, revealed the presence of several nematodes of the genus Philometra Costa, 1845, including three new species: P. austropacifica n. sp. (males and females) from the ovary of Alepes vari (Carangidae), P. piscaria n. sp. (males) from the ovary of Epinephelus coioides (Serranidae), and P. selaris n. sp. (males) probably from the abdominal cavity (found in washings) of Selar crumenophthalmus (Carangidae). The new species are characterised mainly by the length and structure of the spicules and gubernaculum, body size, their location in the host and the type of host. Philometra austropacifica n. sp. is the first known nominal gonad-infecting species of Philometra parasitising a carangid fish. In addition, the gravid female of P. fasciati Moravec & Justine, 2008 from the ovary of Epinephelus fasciatus (Serranidae) is described for the first time. Carangid host fish were identified by both morphology and DNA barcoding.


Introduction
Despite the report of ten species of Philometra Costa, 1845 from marine fishes in New Caledonian waters [17][18][19][20], our knowledge of the fauna of these important parasites in this region remains fragmentary. Recent studies carried out in adjacent waters off southern Indonesia and northern Australia by Moravec et al. [27], Dewi & Palm [2] and Moravec & Diggles [16] revealed the presence of several previously unknown philometrids in marine fishes, which may also occur in the New Caledonia region. Additional samples of philometrid nematodes, including three new species of Philometra, were collected from carangid and serranid perciform fishes. A taxonomic evaluation of this material is presented herein.

Materials and methods Fish
Fishes were caught by line, speared or bought from the fish market; carangids from the fish market were taken with mackerel nets within a few miles off Nouméa, New Caledonia and were very fresh. In all cases the fishes came from locations within 50 km of Nouméa. All fish specimens were measured, weighed and photographed (except for Epinephelus coioides, see below). A unique number (JNC) was assigned to each fish. The parasitological material was then assigned a corresponding JNC linked to the respective fish host. Philometrid nematodes used in this study were recorded from the following four species of New Caledonian fishes: Carangidae: the herring scad, Alepes vari (Cuvier), and the bigeye scad, Selar crumenophthalmus (Bloch); Serranidae: the orange-spotted grouper, Epinephelus coioides (Hamilton), and the blacktip grouper, E. fasciatus (Forsskål). Since morphological identification of carangids is sometimes difficult, identification was confirmed by DNA barcoding. For E. coioides, we collected the material from fish ovaries sold at the fish market, other parts of the fish being sold separately. A discussion with the seller provided a reasonable certainty that all of the ovaries were from the same species; however, identification could not be ascertained by a direct examination of the fish.

Molecular identification of fish
Fish DNA was extracted from tissue samples of three specimens using the NucleoSpin 96 tissue kit (Macherey-Nagel) following the manufacturer's instructions. The 5 0 region of the cytochrome oxidase I (COI) mitochondrial gene was amplified using the primers FishF1 (5 0 -TCAACCAACCACAAAGA CATTGGCAC-3 0 ) and FishR1 (5 0 -TAGACTTCTGGGTGGC CAAAGAATCA-3 0 ) (Ward et al. 2005 [36]). The PCR reactions were performed in a 20-lL solution, containing 1 ng of DNA, 1 · CoralLoad PCR buffer, 3 mM MgCl2, 66 lM of each dNTP, 0.15 lM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was: 4 min at 94°C, followed by 40 cycles of 94°C for 30 s, 48°C for 40 s, 72°C for 50 s, with a final extension at 72°C for 7 min. PCR products were purified and sequenced in both directions on 3730xl DNA Analyser 96-capillary sequencer (Applied Biosystems). 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 KJ192344, KJ192345 and KJ192346. Species identification was confirmed using the BOLD identification engine [35]. The fish nomenclature adopted follows FishBase [3].

Parasites
Nematodes were collected from fish examined with a wash method for intestines [6,7]. Fish ovaries were separated, examined for large philometrid females, and scraped to look for smaller males.
The nematodes for morphological studies were fixed in hot 4% formaldehyde solution in physiological saline, or sometimes in hot 70% ethanol. For light microscopic examination, they were cleared with glycerine. Drawings were made with the aid of a Zeiss microscope drawing attachment. Specimens used for 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. Etymology: The specific name of this nematode is a Latin adjective composed of the words australis (= southern) and Pacificus (= Pacific), which relates to the region of the occurrence of this parasite, that is South Pacific.

Description
Male (eight specimens; measurements of holotype in parentheses): body whitish, filiform, tapering to both ends, 1.47-2.73 (2.73) mm long; maximum width at middle 48-69 (69); anterior part of body slightly narrower just posterior to cephalic end (Fig. 1D); body width at this narrowed part 27-36 (33). Maximum width/body length 1:27-43 (1:40); width of cephalic end 33-39 (36), that of posterior end 30-33 (36). Cuticle smooth. Cephalic end rounded. Oral aperture small, oval, surrounded by 14 cephalic papillae arranged in two circles: external circle formed by four submedian pairs of papillae (each pair consisting of one circular and one narrower, more elongate papilla); internal circle formed by four submedian and two lat-   Subgravid female (two small complete specimens and four body fragments of larger specimens; measurements of allotype in parentheses): body of fixed specimens whitish to brownish, filiform, with rounded ends; posterior part of body narrower than anterior part. Body length of complete specimens 9.

Discussion
Of the 16 gonad-infecting species of Philometra described with lamella-like structures on the gubernaculum [13], a distinct dorsal protuberance near the distal extremity of the gubernaculum, which occurs in the new species, is only present in P. gerrei [10,13,14,19,22,23,32]. However, the protuberance consisting of two dorsolateral lamellar parts separated from each other by a smooth median field, as shown to occur in the new species, is known only in P. johnii, from Johnius dussumieri (Cuvier) (Sciaenidae) in the Persian Gulf and off northern Australia [10,16], P. otolithi, from Otolithes ruber (Bloch & Schneider) (Sciaenidae) in the Persian Gulf, the Sea of Oman and off India [21][22][23], and P. lopholatili, from Lopholatilus chamaeleonticeps Goode & Bean (Malacanthidae) in the Gulf of Mexico [13].
The morphology of all these three species is similar to that of P. austropacifica n. sp. However, in contrast to the new species, the male caudal mound of P. johnii and P. otolithi is distinctly dorsally interrupted (vs. non-interrupted) and the distal lamellar tip of their gubernaculum is somewhat broader and shorter, with lamellae extending posteriorly to the smooth dorsal field and almost to the gubernaculum extremity (vs. lamellae extending posteriorly only as far as the level of the end of the smooth field). They also differ in the host family (Sciaenidae vs. Carangidae).
The shape of the male caudal mound and the structure of the distal tip of the gubernaculum in P. lopholatili are similar to those of the new species, but the males of P.  [9]. Of the eight identified philometrid species reported from carangid fishes, only two, Philometra globiceps (Rudolphi, 1819) and P. lateolabracis (Yamaguti, 1935), were found in the gonads of Alepes djedaba (Forsskål) (as Caranx kalla) and Seriola dumerili (Risso), respectively [9,31]; other species are found in the abdominal cavity, eyes, musculature or subcutaneous tissues of their fish hosts. However, as pointed out by Quiazon et al. [33] and Moravec & Ali [10], these two species were evidently misidentified. Consequently, P. austropacifica n. sp. is the first known nominal gonad-infecting species of Philometra parasitising a carangid fish.
Site of infection: Ovary. Type locality: Fish market, Nouméa City, New Caledonia. Date: 30 October 2009. Prevalence and intensity: Prevalence unknown; five nematode specimens. Twenty whole ovaries were examined at the fish market the same day and female philometrids were visible in none of them. Male philometrids were found in 1/1 ovary macerated and examined under a binocular microscope.
Type specimens: Holotype and one paratype in the Muséum National d'Histoire Naturelle, Paris (MNHN JNC 3082) and one paratype in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Č eské Budějovice (Cat. No. N-1040).
Etymology: The specific name piscaria is an adjective derived from the Latin name for a fish market, that is (taverna) piscaria, and relates to the place where specimens of this nematode species were obtained.     [13,15]. Two of them, P. cyanopodi from Epinephelus cyanopodus (Richardson) and P. fasciati from E. fasciatus (Forsskål), were described from New Caledonian waters [17,18].
Two other philometrid species, Philometra epinepheli Dewi & Palm, 2013 and Spirophilometra endangae Dewi & Palm, 2013, were recently described based on females found in the opercula and fins, respectively, of E. coioides in the South Bali Sea, Indonesia [2]. Subsequently, the same two species were recorded in E. coioides off the northern coast of Australia [16], so it is highly probable that they also occur in this fish host in New Caledonian waters. Since the male of P. epinepheli remains unknown, as does the female of P. piscaria, for the time being it is impossible to distinguish these two species morphologically. However, individual species of Philometra are known to be characterised by their location in the host [5,9], so these two species can be distinguished on the basis of their very different sites of infection (ovary vs. fins).

Description
Gravid female (one specimen): body of fixed specimen brownish (red when alive), with distinct dark-coloured intestine visible through cuticle, with rounded ends. Posterior part of body narrower than anterior part; maximum width in region posterior to oesophagus. Body 382 mm long, maximum width 1.63 mm, maximum width/body length ratio 1:234. Width of cephalic end 218. Cephalic papillae very small, indistinct when viewed laterally (Fig. 5A). Oral aperture large, oval, surrounded by four submedian double cephalic papillae of external circle and six single papillae (two lateral and four submedian) of internal circle. Amphids indistinct. Base of mouth formed by lobes of three oesophageal sectors (Figs. 5B, 6A, B). Oesophagus including anterior bulbous inflation 1.58 mm long, representing 0.4% of body length; bulb well developed, 231 long and 231 wide; maximum width of oesophagus including gland 204. Oesophageal gland well developed, opens into oesophagus just posterior to nerve ring, with large cell nucleus in middle. Nerve ring and oesophageal nucleus 340 and 952, respectively, from anterior extremity. Ventriculus indistinct. Intestine wide at anterior end; its posterior end narrow, attached by short ligament ventrally to body wall near caudal end; ligament 544 long. Vulva and anus absent. Ovaries relatively short, thick, reflected, situated near body ends. Uterus occupies most of body space, reaching to level of nerve ring, filled with numerous larvae 369-561 long and 21-27 wide; length of oesophagus 126-180, of tail 69-102, representing 32-36% and 17-19%, respectively, of entire body length of larva (Fig. 5C). Posterior end of female narrows abruptly in region of ligament, rounded, 204 wide, with 2 minute, indistinct subterminal papilla-like projections (Figs. 5D, E).

Discussion
Philometra fasciati was established by Moravec & Justine [18] based on their earlier description of the male nematodes found in the ovary of Epinephelus fasciatus off New Caledonia and originally considered to be P. lateolabracis [17]. The correct identification of these nematodes was only possible after the description of the previously unknown male of P. lateolabracis by Quiazon et al. [32].
As mentioned above, P. fasciati is known only from males and a single small mature female 3.16 mm long [17]. The present description of the conspecific gravid female extends our knowledge of the morphology of this species considerably. Among gonad-infecting species of Philometra parasitising serranid fishes, which have gravid females of <200 mm in length in most species, P. fasciati is the second largest (384 mm), being longer (445 mm) only in material identified as P. managatuwo by Moravec et al. [26] from Epinephelus septemfasciatus (Thunberg) off Japan. The gravid female of P. fasciati differs from those of other gonad-infecting species of Philometra in that its paired submedian cephalic papillae of the external circle are fused together to form large, somewhat elevated double papillae, in contrast to the well-separated papillae of other species.
Site of infection: Probably abdominal cavity (found in wash).
Type locality: Fish market, Nouméa City, New Caledonia. Fish were taken with mackerel nets within a few miles off Nouméa. Dates: 10 September 2009 and 25 November 2009. Prevalence and intensity: two fish infected/seven fish examined using the ''wash'' method; one nematode specimen per fish.
Type specimen: Holotype (mounted on SEM stub) in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Č eské Budějovice (Cat. No. N-1043). The paratype specimen was destroyed during the SEM procedure.
Etymology: The specific name of this nematode relates to the genitive form of the generic name of the host.

Discussion
The only philometrid species so far reported from S. crumenophthalmus is Philometroides atropi (Parukhin, 1966). It was originally described by Parukhin [29] based solely on females found in the abdominal cavity of the carangid fish Atropus atropos (Bloch & Schneider) off Vietnam (South China Sea, Gulf of Tonkin), and later the same author [30] reported it from the stomach wall of S. crumenophthalmus in the Red Sea. However, considering a relatively high degree of host specificity of philometrid nematodes, differences in the site of infection in the host and the fact that only nematode females were studied, it may well be that nematodes from S. crumenophthalmus were not conspecific with those from P. atropi. Since Parukhin's material is probably lost (personal communication with Prof. A.V. Gaevskaya, Sevastopol), its reexamination is no longer possible.
Most of these six species are known solely from their females, whereas conspecific males have been described only for C. americanum and P. tauridica [5,12]. In contrast to both of these species, philometrid males in the present material from S. crumenophthalmus are markedly larger (body length 5.3-5.5 vs. 3.1-3.3 or 1.5-3.0 mm) and their gubernaculum is also longer (72-75 vs. 48-51 or 30-58 lm); moreover, they were collected from fishes belonging to a different genus (Selar vs. Caranx or Trachurus) in a distant geographical region (South Pacific vs. North Atlantic or Mediterranean region). Males of the remaining four philometrid species from carangids are not known and, consequently, cannot be compared with those of the present material; however, these species can be separated based on the different genus of their type host and the geographical distribution (see above). Therefore, we consider the two philometrid males from S. crumenophthalmus to represent a new species, P. selaris n. sp. The allocation of this species to Philometra is provisional; present philometrid genera are mostly based on the morphology of gravid and subgravid females [1,5,9,15,34], whereas males of some genera (e.g. Caranginema, Philometra and Philometroides) are unidentifiable to the generic level.