Two gonad-infecting species of Philometra (Nematoda: Philometridae) from marine fishes off the northern coast of Australia

Two different gonad-infecting species of Philometra Costa, 1845 were collected from the ovary of marine perciform fishes, the blackspotted croaker Protonibea diacanthus (Sciaenidae) and the John’s snapper Lutjanus johnii (Lutjanidae), from off the northern coast of Australia. Nematodes (males and females) from P. diacanthus represent a new taxon, Philometra protonibeae n. sp., which is mainly characterized by the body length of the males (3.37–3. 90 mm), broad, equally long spicules (length 126–141 μm) and the shape and structure of the gubernaculum with a dorsally lamellate distal tip. The nematodes (only females) from L. johnii may represent an undescribed species, but, because of the absence of conspecific males, they could not be specifically identified. Philometra protonibeae is the fifth nominal gonad-infecting species of this genus recorded from marine fishes in Australian waters and the seventh species of these parasites described from fishes of the family Sciaenidae.


Introduction
The knowledge of the fauna of philometrid nematodes (Philometridae) parasitizing fishes in Australian waters remains fragmentary. To date, a total of 16 valid nominal species of these parasites belonging to the genera Buckleyella Rasheed, 1963 (1 species), Philometra Costa, 1845 (11 species), Philometroides Yamaguti, 1935 (3 species) and Spirophilometra Parukhin, 1971 (1 species) have been recorded in this region [3, 14-16, 28, 32]. An additional 13 nominal species of Philometra parasitizing marine fishes were reported from off New Caledonia, South Pacific [12,[18][19][20][21], which may also occur in Australian waters. Beveridge et al. [1] have recently reported a 45% overall similarity between the fauna of trypanorhynch metacestodes in teleosts of the Great Barrier Reef and New Caledonia and a similar situation may occur in the case of philometrids.
During recent helminthological investigations of some marine fishes off the northern Australian coast, philometrid nematodes were collected from the ovary of two species of perciform fishes, the blackspotted croaker Protonibea diacanthus (Lacépède) (Sciaenidae) and the John's snapper Lutjanus johnii (Bloch) (Lutjanidae). A close examination revealed that they represent two different species of Philometra, one from the former host, which represents a new species, and the other from the latter host which cannot be identified to species due to the absence of the males. Both of these forms are described herein. The two aforementioned hosts are tropical marine fishes with an Indo-West Pacific distribution and are targeted by commercial and recreational fishermen [2].

Materials and methods
Fish were collected by hook and line from six locations along the northern Australia coastline (Table 1). All fish were anaesthetized, killed and stored in ice for transportation back to the laboratory for further examination. At the laboratory, each fish was measured (total length; mm), the body cavity opened and all internal organs removed. Internal organs were placed in a sealed plastic bag and frozen until examined for parasites. At this time, the ovaries were separated from the remaining internal organs, placed in a Petri dish of physiological saline and opened under a dissecting microscope and examined for parasites.
The nematodes obtained were washed in physiological saline and were then fixed and preserved in 70% ethanol. For light microscopical examination, the nematodes were cleared with glycerine. Drawings were made with the aid of a Zeiss drawing attachment. Specimens used for scanning electron microscopy (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. The fish nomenclature adopted follows FishBase [2]. Etymology: The specific name of this nematode relates to the genitive form of the generic name of the host.
All of the above-mentioned valid species are easily distinguishable from P. protonibeae n. sp. by the morphology of the males and, in particular, the structure of the distal end of the gubernaculum. In contrast to the new species, the gubernaculum of P. carolinensis and P. floridensis is smooth, not transversely lamellate, but possesses a distinct dorsal barb on its distal end. The gubernaculum of P. johnii, P. otolithi and P. sciaenae is provided with many marked transverse lamella-like structures forming a distinct dorsal protuberance visible in lateral view, as in the new species. However, the dorsal protuberance on the gubernaculum of P. johnii and P. otolithi appears to be single in lateral view but in fact consists of two dorsolateral parts separated from each other by a smooth longitudinal field when observed dorsally. Quiazon et al. [31] illustrated a bipartite dorsal protuberance on the gubernaculum of P. sciaenae, where no median smooth field between the two parts of the protuberance was present, but the lamella-like structures on the protuberance were interrupted dorsally by a median longitudinal line. In contrast to P. johnii, P. otolithi and P. sciaenae, the protuberance on the gubernaculum of P. protonibeae is simple, without a division into two parts, and its lamella-like structures are not interrupted dorsally.
As mentioned above, P. lateolabracis has previously been reported from the gonads of Protonibea diacanthus off India [4]. Since only specifically unidentifiable females were available, it may well be that they were conspecific with P. protonibeae n. sp. Although the male morphology of P. lateolabracis, as redescribed by Quiazon et al. [30] based on specimens from the type host, is similar to that of P. protonibeae and these two species resemble each other in the lengths of spicules and gubernaculum (65-130 and 50-93 lm in P. lateolabracis vs. 126-141 and 78-90 lm in P. protonibeae), the males of the new species are somewhat longer (3.37-3.90 mm vs. 2.07-2.73 mm). However, the two species differ distinctly in the structure of the gubernaculum: in P. protonibeae the narrowed distal tip posterior to the dorsal protuberance is markedly shorter and transversely lamellate (vs. elongate, approximately as long as the protuberance and without transverse lamellae); the dorsally uninterrupted transverse lamellae extend anteriorly only to the proximal end of the dorsal protuberance (vs. extending anteriorly into the region anterior to the protuberance); and a few (two or three) oblique lamellae and a small median triangular structure are present at the dorsal region anterior to the proximal end of the protuberance (Figs. 1K, 2E) (no such structures were observed in P. lateolabracis, but the gubernaculum of this species has not yet been studied in dorsal view). The abovementioned triangular dorsal structure on the gubernaculum has not been described for any other species of Philometra. Philometra protonibeae and P. lateolabracis also differ in the shape of the distal regions of the spicules, which are markedly broad (c. 4 lm) in lateral view in the former species, but relatively narrow (c. 2 lm wide) in lateral view in the latter species.
Females of Philometra lateolabracis have recently been reported by Sethi et al. [33] from the caudal fins of two sciaenid fishes, Otolithes ruber (Bloch et Schneider) and Pennahia macrophthalmus (Bleeker) (= P. anea (Bloch)), in the Bay of Bengal, off India. However, this is an evident misidentification. As can be seen in the published photographs of the worms from O. ruber, the structure of the oesophagus (presence of a conspicuously large anterior oesophageal bulb) and the presence of marked cuticular ornamentations on the body indicate that these nematodes, at least those from O. ruber, represent the philometrid Clavinemoides annulatus Moravec, Khosheghbal & Pazooki, 2013, a species recently described from the caudal fin of the same host species (O. ruber) in the Persian Gulf off Iran [22].
All of the above-mentioned species are distinguishable from each other mainly on the basis of male morphology (in fact, P. mira and P. synagridis are known only by their males), whereas the morphology of conspecific gravid and subgravid females is rather uniform, not enabling reliable species identification. The presence of fairly large, papilla-like caudal projections and a well-developed anterior oesophageal bulb in gravid females of the present material from L. johnii indicates that these nematodes may belong to an undescribed species (caudal projections are missing or much smaller in other species parasitizing Lutjanus spp. and their oesophageal bulb is usually less developed). However, in view of the absence of males and the high degree of host specificity in gonad-infecting species of Philometra when different species are found in congeneric fish hosts in the same locality [10,25,26], we refrain from establishing a new species for these nematodes until conspecific males are available.
Kardousha [5] reported philometrid females identified as Philometra lateolabracis from the ovary of the same host species, L. johnii, in the Persian Gulf, but in view of the revisionary paper of Quiazon et al. [30] on P. lateolabracis, this would appear to be a misidentification.