Two gonad-infecting species of Philometra (Nematoda: Philometridae) from groupers (Serranidae) off Tunisia, with a key to Philometra species infecting serranid gonads

Based on light and scanning electron microscopical studies of nematode specimens (males and mature females) collected from the ovary of groupers (Serranidae, Perciformes) in the Mediterranean Sea off Tunisia (near Tunis and Sfax), two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) are reported: Philometra inexpectata n. sp. from the mottled grouper Mycteroperca rubra and P. jordanoi (López-Neyra, 1951) from the dusky grouper Epinephelus marginatus. Identification of both fish species was confirmed by molecular barcoding. The new species is mainly characterized by the length of equally long spicules (147–165 μm), the gubernaculum (63–93 μm long) bearing at the tip two dorsolateral lamellar parts separated from each other by a smooth median field, a V-shaped mound on the male caudal extremity, the presence of a pair of large caudal papillae located posterior to the cloaca and by the body length of the males (1.97–2.43 mm). Philometra inexpectata n. sp. is the fifth known gonad-infecting philometrid species parasitizing serranid fishes in the Mediterranean region. The males of P. jordanoi were examined by scanning electron microscopy for the first time; this detailed study revealed some new taxonomically important morphological features, such as the number and arrangement of cephalic and caudal papillae, presence of amphids and phasmids and mainly the lamellate structures at the posterior end of the gubernaculum. A key to gonad-infecting species of Philometra parasitic in serranid fishes is provided.


Introduction
Gonad-infecting species of philometrid nematodes (Philometridae) are widely distributed in marine fishes of the Atlantic, Indian and Pacific Oceans, and sometimes occur in brackish-water environments [21,26]. These parasites may be severely pathogenic in fish ovaries and can affect reproduction [15].
The species identification of these parasites, previously mostly based on the morphology of large-sized females, was rather problematic. However, scanning electron microscopical (SEM) examinations of minute philometrid males made the identification more reliable and indicated considerable species diversity in these nematodes. To date, many gonad-infecting species of Philometra Costa, 1845 have been described from a variety of marine fishes belonging to different families and their number is quickly increasing [13,14,16,17,19,20,22,23,[27][28][29].
During recent helminthological investigations of some marine fishes in the Mediterranean Sea off the Tunisian coast near Tunis and Sfax [2,28], males and mature females of philometrid nematodes were collected from the ovary of two species of serranid fishes, the mottled grouper Mycteroperca rubra (Bloch) and the dusky grouper Epinephelus marginatus (Lowe) (both Serranidae, Perciformes). A close examination revealed that they represent one new and one known insufficiently studied species. Both host species are subtropical marine fishes, which are distributed in the Mediterranean Sea and the eastern Atlantic (M. rubra) or in the eastern and southern Atlantic and western Indian Oceans (E. marginatus) and are targeted by commercial and recreational fisheries [3].

Fish and their identification
Fish were purchased at the fish market in Tunis and Sfax, Tunisia; these were previously caught by fishermen in the nearby coastal waters of the Mediterranean Sea. Fish DNA was extracted from tissue samples using the NucleoSpin 96 tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Sequences were obtained by amplification and sequencing of a region of the cytochrome oxidase subunit I (COI) mitochondrial gene using the primers FishF1 (5 0 -TCAACYAATCAYAAAATYGGCAC-3 0 ) and FishR1 (5 0 -TGATTYTTYGGYCACCCRGAAGT-3 0 ) [34]. Standard PCRs were carried out in 20 lL total volume, containing about 30 ng of DNA, 1 · 10· PCR buffer, 2 mM MgCl 2 , 200 lM mix dNTPs, 150 nM of each primer and 1 unit of Taq polymerase (Qiagen, Hilden, Germany).
After an initial denaturation of 3 min at 95°C, the mitochondrial DNA was amplified through 39 cycles of 15 s at 95°C, 20 s at 48°C and 40 s at 72°C, with a terminal elongation for 5 min at 72°C. PCR products were purified and sequenced in both directions on 3730xl DNA Analyser 96-capillary sequencer (Applied Biosystems, Waltham, MA, USA). 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 KU739518-KU739521. Species identification was confirmed using the BOLD identification engine [32]. Since BOLD does not include all sequences available in GenBank but includes others, comments are added for similarities with other sequences. The fish nomenclature adopted follows FishBase [3].

Nematodes
Philometrid specimens were collected from frozen-thawed fish gonads under the dissecting microscope. They were fixed in hot 70% ethanol and cleared with glycerine for light microscopical (LM) examination. Drawings were made with the aid of a Zeiss drawing attachment. Specimens used for scanning electron microscopy 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. Type-host: Mottled grouper, Mycteroperca rubra (Bloch) (Serranidae, Perciformes). After identifying the fish based upon morphological characteristics, identification was confirmed via barcoding. The COI sequence obtained for our specimen (GenBank Accession Number KU739518) was identical (100% similarity, 94% cover) to a sequence identified as M. rubra from off Israel (KF564307; unpublished). BOLD provided similar results. Mycteroperca rubra could be confused with the Island grouper M. fusca (Lowe), a species from the eastern Atlantic Ocean. Heemstra et al. (2010) [5] reported the presence of M. fusca for the first time in the Mediterranean Sea, off Israel. They wrote: ''A reasonable possibility is that M. fusca entered the Mediterranean through the Strait of Gibraltar, as many Atlantic species do, then expanded its distribution along the North African coast and was overlooked or confused with M. rubra.'' Unfortunately, no COI sequence of M. fusca is available for comparison. Incidentally, we also noted that the sequence we obtained from our specimen was very close (99% similarity, 94% cover) to several other sequences labelled as M. acutirostris from Brazil (e.g., KF836485; unpublished); M. acutirostris also resembles M. rubra and M. fusca morphologically, but it has never been reported in the Mediterranean Sea [5]. We thus conclude that the species studied herein from Tunisia is M. rubra, but we note that barcoding of more species is needed.

Results and discussion
Site of infection: Ovary.
Morphological and biometrical differences of P. inexpectata n. sp. from all the above-mentioned species are apparent from the identification key at the end of this paper. To date, only two gonad-infecting species of Philometra have been reported to parasitize hosts of the genus Mycteroperca Gill: P. charlestonensis, a parasite of M. phenax in the North American Atlantic region (USA) [25], and P. lateolabracis (Yamaguti, 1935) from M. rubra in the Mediterranean Sea off Turkey (Iskenderun Bay) [18]. However, with respect to the redescription of P. lateolabracis by Quiazon et al. [30], the nematodes from M. rubra off Turkey, studied only by LM, were evidently misidentified [12]. Since their morphology and measurements agree with those of the newly described species and because the host species (M. rubra), localization in the host and the geographical region (Mediterranean Sea) are identical, they evidently belong to P. inexpectata n. sp.
Philometra charlestonensis distinctly differs from P. inexpectata n. sp. in having shorter spicules (123-141 lm vs. 147-165 lm) and mainly in the male caudal mound consisting of two lateral parts widely separated from each other dorsally (vs. caudal mound V-shaped, not interrupted dorsally).
Material from Tunisia. Host: Epinephelus marginatus. After identifying the fish based upon morphological characteristics, identification was confirmed via barcoding. We obtained sequences (GenBank KU739519-KU739521) from three specimens; only one (corresponding sequence: KU739521) had mature gonads and was used in this study. The three sequences were almost identical, with 1-2 bp differences; BLAST results show that our sequences were identical or almost identical (99-100% similarity) to several sequences all identified as E. marginatus, such as KC500679 (from Iskenderun Bay, Turkey [7]), JF493449 and JF493450 (from South Africa, unpublished), KM077929 (Senegal [33]) and sequences from Brazil such as KF836469 (unpublished). Our sequences were also identical or almost identical (99-100% similarity) to shorter sequences identified as E. marginatus such as FN688939 (from France [8]). BOLD gave similar results, but the automatic identification was disrupted by the presence in the database of an ''early sequence'' labelled as Epinephelus fasciatus (Forsskål) (clearly a misidentification). We consider that barcoding definitely demonstrates that our specimens belong to E. marginatus.

Remarks
This species was originally described by López-Neyra [9] as Sanguinofilaria jordanoi, based solely on female specimens collected in the ovary of E. marginatus [syn. E. gigas (Brünnich)] obtained from the market in Tétouan, Morocco (probably caught in the Mediterranean Sea). Later the species was transferred to Philometra as P. jordanoi [36]. The genus Sanguinofilaria Yamaguti, 1941 was subsequently synonymized with Philometra [31]. The original description of P. jordanoi [9] was inadequate. Later Moravec et al. [24], based on available nematode specimens from the ovary of wild E. marginatus collected in the Mediterranean Sea near the Balearic Islands, Spain and those from wild and cultured E. marginatus in the Tyrrhenian Sea off Sicily, Italy, provided a somewhat more detailed description of philometrid gravid females, which was, more or less, in agreement with that of P. jordanoi. Nevertheless, the authors identified this material as P. lateolabracis, a species described from females collected in three species of perciform fishes off Japan [35], and designated P. jordanoi to be its junior synonym.
Subsequently, Moravec and Genc [18], based on available body fragments of nematode gravid females from the ovary of E. marginatus in the Mediterranean Sea off Turkey (Iskenderun Bay), described the female anterior end including cephalic structures of these nematodes and the first-stage larva from the uterus. They again identified these nematodes as P. lateolabracis. From the same locality (Iskenderun Bay off Turkey) and the same host species, P. lateolabracis was also reported by Genc et al. [4].
Merella et al. [10] were the first to provide the description of the male (based on a single available specimen studied by LM) of a philometrid collected from the ovary of E. marginatus in waters near Majorca, Spain, which was identified as P. lateolabracis. But in their subsequent paper [11], they re-erected P. jordanoi, to which they assigned the abovementioned male specimen.
However, Quiazon et al. [30] were the only ones to discover the males of P. lateolabracis from the type-host in Japan and provided their detailed description based on LM and SEM examinations, which enabled a comparison with other gonadinfecting Philometra spp. with described males. Their study showed that P. lateolabracis is a specific parasite of Lateolabrax japonicus (Cuvier) (Lateolabracidae) and that all previous records of this parasite from many other fish species were apparently based on misidentifications. Comparison of the males of P. lateolabracis and those of P. jordanoi confirmed the validity of the latter species [12].
It is apparent that all the previous records of P. lateolabracis in E. marginatus in the Mediterranean region [4,18,24] concerned, in fact, P. jordanoi. Also, the nematodes designated as Philometra sp. from the ovary of E. marginatus in Iskenderun Bay off Turkey [1] should be assigned to this species.
The present detailed study of the males of P. jordanoi, including the first use of SEM, made it possible to describe some new taxonomically important morphological features in this species, such as the presence of lamellate structures on the distal end of the gubernaculum, the number and distribution of cephalic and caudal papillae, the character of the male caudal mound, the structure of the male oesophagus, the location of the nerve ring and excretory pore, and the morphology of the mature female. As compared with other gonad-infecting congeneric species parasitizing serranid fishes, P. jordanoi is remarkable for its rather long spicules, which may attain up to 265 lm [11]. The comparison of P. jordanoi with other species from the gonads of serranids is more apparent from the following key.
Key to gonad-infecting species of Philometra parasitizing fishes of the family Serranidae