Species of Philometra (Nematoda, Philometridae) from fishes off the Mediterranean coast of Africa, with a description of Philometra rara n. sp. from Hyporthodus haifensis and a molecular analysis of Philometra saltatrix from Pomatomus saltatrix

Two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) were recorded for the first time from marine perciform fishes off Tunisia and Libya: Philometra rara n. sp. from the rare, deep-water Haifa grouper Hyporthodus haifensis (Serranidae) off Libya and Philometra saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Pomatomidae) off Tunisia. Identification of both fish species was confirmed by molecular barcoding. Light and scanning electron microscope studies of Ph. rara n. sp. showed that it is characterized by the length of spicules (216–219 μm) and the gubernaculum (90–93 μm), the gubernaculum/spicules length ratio (1:2.32–2.43), and mainly by the shape and structure of the distal end of the gubernaculum (shovel-shaped with a wide median smooth field in dorsal view), appearing as having a dorsal protuberance in lateral view, and by the structure of the male caudal mound (dorsally interrupted); large subgravid females (70–137 mm long) are characterized by the presence of four oval submedian cephalic elevations, each of them bearing a pair of cephalic papillae of the outer circle. The finding of Ph. saltatrix off Tunisia confirms that this species is widespread throughout the Mediterranean region. A molecular analysis of our Ph. saltatrix specimens and other available philometrid cytochrome c oxidase 1 (COI) sequences showed that most species have robust clades. Sequences of Ph. saltatrix from Tunisia diverge from Ph. saltatrix from Brazil and the USA, suggesting that speciation is currently occurring between populations from both sides of the Atlantic Ocean.

. During recent helminthological investigations of some marine fishes in the Mediterranean Sea off the coast of Libya and Tunisia, an additional two species of Philometra were recorded, one known and one new to science. Results of their study are presented herein.
The Haifa grouper Hyporthodus haifensis (maximum body length 110 cm, maximum weight 25 kg) is a deep-water subtropical fish distributed in the Eastern Atlantic (Mediterranean to southern Angola) [6]. Other parasites (diplectanid monogeneans) were recently reported from H. haifensis from the same locality [1]. The bluefish Pomatomus saltatrix (maximum body length 130 cm, weight up to 14.4 kg) is an important commercial fish and game fish with a circumglobal distribution in tropical and subtropical waters [6].
Molecular analysis of Philometra species has concerned only a small number of species [4,28,33,34]; sequences of cytochrome c oxidase 1 (COI), typically used for barcoding in other animals [30], were demonstrated to be effective in identifying philometrid species [4,34]. In our study, we obtained sequences of COI from several Ph. saltatrix specimens and compared them with available sequences from other species.

Fish and their identification
Fish were purchased at fish markets in Tunis and Sfax, Tunisia; these were previously caught by fishermen in the nearby coastal waters of the Mediterranean Sea.
A single, whole specimen of Haifa grouper, Hyporthodus haifensis (Ben-Tuvia) (Serranidae), with mature gonads ( Fig. 1) was caught from off Libya according to fishmongers of the Sfax fishmarket, Tunisia. Identification of the fish was performed according to usual keys and books [6,12] and was confirmed by barcoding.

Barcoding of fish
Fish DNA was extracted from tissue (gonad) 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 c oxidase subunit I (COI) mitochondrial gene using the primers FishF1 (5'-TCAA-CYAATCAYAAAATYGGCAC-3') and FishR1 (5'-TGAT-TYTTYGGYCACCCRGAAGT-3') [35]. Standard Polymerase chain reactions (PCRs) were carried out in 20 lL total volume, containing about 30 ng of DNA, 1 · 10x 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, amplification was performed 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 Analyzer 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 KY500054-KY500065. Species identification was confirmed using the BOLD identification engine [30]. Since BOLD does not include all sequences available in GenBank but includes others, comments are added for similarities with other sequences.

Collection of philometrids
Philometrid specimens were collected from fresh (Haifa grouper) or frozen-thawed (Bluefish) gonads under the dissecting microscope. They were fixed in hot 70% ethanol and processed for examination or molecular techniques.

Barcoding of philometrids
The same method was used for female fragments (ca. 1-2 mm 3 ) and individual whole males. Genomic DNA was extracted using the QIAamp DNA Micro Kit (Qiagen).
A % 400 bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene was amplified with the nematode-specific PCR primers NemCOI5P (CATTTRTTTTGRTTTTTTGG) and NemCOI3P (ACYACATRATAAGTATCRTG) [4]. PCRs were performed in a final volume of 20 lL, containing 1.5 lL isolated DNA, 1 · CoralLoad PCR buffer, 3 mm

Trees and distances
A tree was constructed from most available COI sequences of philometrids, including sequences already available in GenBank and our new sequences. The analysis involved 36 nucleotide sequences and there were a total of 234 positions in the final dataset. The tree, computed in MEGA7 [9] with 1000 bootstrap replications [5], was inferred using the Neighbour-Joining method [32] and Kimura-2 parameter distance [8]. Clavinema sp. was set as the outgroup. Genetic distances (Kimura-2 parameter distance [8]) were estimated with MEGA7 [9]. All codon positions were used.

Description of philometrids
Philometrid specimens were cleared with glycerine for light microscope (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 (Gentle Beam (GB) low mode). All measurements are in micrometers unless indicated otherwise.

Molecular study of host fish
Haifa grouper, Hyporthodus haifensis The sequence of our specimen (KY500054), submitted to a BLAST in GenBank, showed 100% similarity with three sequences of the same species collected off Libya and Tunisia [1], and, in BOLD [30], showed 100% similarity with sequences of the same species from off Sicilia, Italy [10]. This confirms the identification of the species.

Bluefish, Pomatomus saltatrix
Whole specimens of Bluefish with mature gonads were purchased and were barcoded. When asked for more mature gonads showing visible philometrid females, fishmongers of the Tunis fishmarket were keen to provide to one of us (AC) selected infected gonads, allegedly from several fish species. The day after, the gonads were examined at the fishmarket, chosen for the presence of female philometrids and purchased, but they were sold separated from the fish; they were processed for parasitological examination and a tissue sample was taken for each gonad. After barcoding (Table 1), it was found that all fish gonads in fact belonged to a single species, Pomatomus saltatrix.
Prevalence and intensity: 1 fish infected/1 fish with mature gonads examined; 4 nematode specimens per fish.
Etymology: The specific name rara is a Latin adjective (rarus = rare, seldom) and relates to a rather rare occurrence of its fish host.

Remarks
Gonad-infecting species of Philometra are known to exhibit a high degree of host specificity [20,21,23,26,27].     [26] have recently published the key to all these gonad-infecting species of Philometra parasitizing serranid fishes, based on morphological and biometrical features. According to the key, Ph. rara n. sp. is closest to Ph. jordanoi, a specific parasite of dusky grouper Epinephelus marginatus (Lowe) in the Mediterranean region [11,13,14,18,24,26]; the male of Ph. jordanoi was redescribed in detail by Moravec et al. [26]. Both these species resemble each other by the length of spicules (216-219 lm in Ph. rara, 213-252 lm in Ph. jordanoi), the number and distribution of male caudal papillae, the approximate number (about 30) of transverse lamellar structures on the gubernaculum distal tip and by the ventral surface of the posterior end of the gubernaculum with two ventral longitudinal grooves. However, Ph. rara differs distinctly from Ph. jordanoi in the shape and structure of the distal end of the gubernaculum (shovel-shaped with a wide median smooth field vs. tongue-shaped with a narrow smooth field in dorsal view; appearing as having a distinct dorsal protuberance vs. without such a protuberance in lateral view). Whereas the caudal mound of Ph. rara is dorsally interrupted, that of Ph. jordanoi is V-shaped, dorsally uninterrupted. The former species also differs somewhat from the latter in the male body length (2.19-2.27 mm vs. 2.45-2.91 mm), the presence (vs. absence) of a body constriction just behind the male cephalic extremity, the length of the gubernaculum (90-93 lm vs. 81-84 lm), the gubernaculum/spicules length ratio (1:2.32-2.43 vs. 1:2.61-3.11) and the relative length of spicules to the length of the body (10% vs. 8%).
The outer cephalic papillae of large females of Ph. rara are located on four distinct submedian cuticular elevations as well as in Ph. jordanoi (as described by Moravec et al., 2003 [24]) or, for example, in Ph. fasciati (reported by Moravec and Justine, 2014 [19]). However, each pair of these papillae is formed by one circular and one elongate-oval papilla in apical view in Ph. rara, in contrast to that of Ph. jordanoi or Ph. fasciati, in which the pair is formed by two circular papillae in apical view. The female caudal end of Ph. rara bears a pair of small lateral papilla-like projections, like e.g. that of Ph. fasciati, but no female caudal projections were reported for Ph. jordanoi.
To date, only one gonad-infecting species of Philometra is known to parasitize hosts of the genus Hyporthodus Gill: Ph. hyporthodi, a parasite of H. flavolimbatus (Poey) in the northern Gulf of Mexico [15]. However, this species can be easily distinguished from Ph. rara n. sp. by the distinctly longer males (3.62-4.07 mm vs. 2.19-2.27 mm), shorter spicules (135-138 lm vs. 216-219 lm) comprising 4% (vs. 10%) of the body length, the caudal mound consisting of two lateral parts widely separated dorsally from each other and by the different shape and structure of the distal end of the gubernaculum. The female caudal projections are absent (vs. present).

Remarks about morphology
The present material consisted mostly of nematode males, whereas nongravid and subgravid (ovigerous) females (maximum body length of about 80 mm) were much less numerous; no gravid (larvigerous) females were present. The morphology of available specimens was in full agreement with the redescriptions of Ph. saltatrix provided by Moravec et al., 2008 [25] and Moravec and de Buron [16].
Philometra saltatrix is a parasite of the bluefish Po. saltatrix, from which it was reported in North America (Northwest Atlantic, USA: off New York, North Carolina and South Carolina) [2,3,16,22,29] and in the Mediterranean Sea (Iskenderun Bay off Turkey and Tuscan Sea off Italy) [18,25]. The present record of Ph. saltatrix from Tunisian waters shows that this parasite is widespread in the Mediterranean Sea. Conspecific with Ph. saltatrix is probably also Philometra sp. reported by Rego et al. [31] from Ph. saltatrix off the Brazilian coast (see [16]). Philometra saltatrix appears to be a specific parasite of Pomatomus saltatrix throughout the distribution area of this fish; however, our molecular results suggest that differences exist between specimens from both sides of the Atlantic Ocean.

Molecular study of philometrids
Sequences of Ph. saltatrix were obtained for five female fragments out of five (100% success). No DNA was obtained from three males, probably because males are much smaller; no modification of the protocol was attempted for males.
The tree produced with the Neighbour-Joining (NJ) method (Fig. 5) included 36 sequences, including our five new sequences of Ph. saltatrix from Tunisia. All species of marine Philometra and Philometroides formed a clade (with, however, low support value, 32%) from which the freshwater species Philometroides sanguineus and Clavinema sp. were clearly separated (distances of these two to all others: 15.7-24.2%). Relationships between marine philometrid clades showed low support; however, this is of minor relevance since the purpose of our study was not to produce phylogenies but to identify robust clades and compare them with hypotheses regarding the validity of species. All clades identified in previous studies were robust in the analysis, with bootstrap values ranging from 96 to 100%. These included the four clades from the same fish species, the Southern flounder Paralichthys lethostigma (Philometroides paralichthydis clade ''bones'' and clade ''fins'' and Ph. overstreeti clade ''teeth'' and clade ''groove''), Ph. carolinensis and Ph. lagocephali.
All seven sequences of Ph. saltatrix formed a robust clade (bootstrap 100%); within this clade, the five sequences from worms collected in fishes from Tunisia formed a robust clade (bootstrap 99%) but the two sequences of worms collected in fishes from Brazil and South Carolina were united in a low support clade, separated from the clade of the Tunisian sequences. The distance of the specimen from Brazil to the Tunisian specimens was 4.9-5.9%, of the specimen from South Carolina to Tunisia 6.8-6.8%, and the specimens from Brazil and South Carolina had a 3.5% distance. Distances between Ph. saltatrix (all sequences) and other clades ranged from 14.6 to 20.1%.

Molecular study of philometrids
Our tree (Fig. 4) confirms philometrid clades identified in previous molecular studies, which were performed when a smaller number of species were available. Particularly, the four clades identified by Palesse et al. [28], i.e. Philometroides paralichthydis clade ''bones'' and clade ''fins'' and Ph. overstreeti clade ''teeth'' and clade ''groove'', all from the same fish, the Southern flounder off South Carolina, are recognized. In each of these clades, the distances between members of each clade ranged from 0 to 2.2%. These distances are similar to what is found in Ph. carolinensis (intra-distance 0.4%) and Ph. lagocephali (intra-distance 0.2-2%). All these clades might probably be considered as four separate species, even though the four clades from the Southern flounder have not received separate binomials [28].
Our results for Ph. saltatrix are puzzling. All five specimens from the Tunisian clade show intra-distances from 0.4 to 1.3%, which correspond to the intra-distances found in other species (Table 2); however, distances within all specimens of the species, including specimens from the Western Atlantic and the Mediterranean Sea, show values from 0.4 to 6.8% (Table 2). This suggests that (a) all specimens from Tunisia belong to a single species; and (b) specimens from the Western Atlantic and the Mediterranean might belong to  Figure 5. Tree of philometrids based on COI sequences. The evolutionary history was inferred using the Neighbour-Joining method. Bootstrap test results are shown next to the branches; red: bootstrap for clades corresponding to nominal species or clades within species (for Philometroides paralichthydis and Philometra overstreeti). For Philometra saltatrix, all specimens are within a well-supported clade but specimens from Tunisia are separated from specimens from the Western Atlantic (Brazil and South Carolina). different species. Specimens from both sides of the Atlantic (the Americas vs. the Mediterranean Sea) were from fish of the same species, Pomatomus saltatrix; our results suggest that the geographical populations of the parasite Ph. saltatrix are currently undergoing speciation. However, all specimens of Ph. saltatrix from all localities were grouped in a single clade with high support (100%). Hence, the case of Ph. saltatrix needs further investigation involving more specimens from more localities throughout the range of bluefish, Po. saltatrix. For practical purposes, it remains that sequences of all philometrid species currently available show robust clades; this suggests that the use of barcoding may be effective in identifying philometrid species in the absence of morphological studies. This is of particular importance when only female fragments are available. As already emphasized in 2011 by Palesse et al. [28], the molecular database is still extremely limited (currently nine species or clades) in comparison to the number of philometrid species currently accepted (more than 150 [17]).

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http:// publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jerôme Depaquit.