Rasheedia n. nom. (Nematoda, Physalopteridae) for Bulbocephalus Rasheed, 1966 (a homonym of Bulbocephalus Watson, 1916), with description of Rasheedia heptacanthi n. sp. and R. novaecaledoniensis n. sp. from perciform fishes off New Caledonia

The nematode genus Bulbocephalus Rasheed, 1966 (Nematoda, Physalopteridae) was found to be a homonym of Bulbocephalus Watson, 1916 (Apicomplexa) and, therefore, a new name, Rasheedia n. nom., is proposed to substitute it. Based on light and scanning electron microscope studies of specimens collected from the digestive tract of perciform fishes off New Caledonia, two new species of Rasheedia are described: R. heptacanthi n. sp. from the Cinnabar goatfish Parupeneus heptacanthus (Mullidae) (type host) and Dentex fourmanoiri (Sparidae), and R. novaecaledoniensis n. sp. from the Indian goatfish Parupeneus indicus (Mullidae). These new species are mainly characterized by the number of anterior protrusible oesophageal lobes (two in R. heptacanthi and four in R. novaecaledoniensis), structure of the oesophagus and the lengths of spicules. An amended diagnosis of Rasheedia and a key to species of this genus are provided. Three previously described congeneric species are transferred to Rasheedia as R. deblocki (Le-Van-Hoa, Pham-Ngoc-Khue & Nguyen-Thi-Lien, 1972) n. comb., R. inglisi (Rasheed, 1966) n. comb. and R. pseudupenei (Vassiliadès & Diaw, 1978) n. comb. Cestocephalus Rasheed, 1966 [genus inquirendum], including C. serratus Rasheed, 1966 and C. petterae (Le-Van-Hoa, Pham-Ngoc-Khue & Nguyen-Thi-Lien, 1972) n. comb., should be considered to be separate from Rasheedia. The names Pseudomazzia Bilqees, Ghazi & Haseeb, 2005 and P. macrolabiata Bilqees, Ghazi & Haseeb, 2005, established for a nematode somewhat resembling Rasheedia spp., should be considered nomina dubia. Rasheedia heptacanthi n. sp. and R. novaecaledoniensis n. sp. are the first representatives of the Physalopteridae recorded from fishes in New Caledonian waters.


Introduction
Currently, there are two systems of classification for spirurine nematodes, one based principally on molecular data [7] and another based on morphological and biological data [3,5,6]. Nevertheless, since the former system seems to be premature at present [12], the suborder Spirurina of the order Spirurida of the latter system is followed in this paper. Of the ten superfamilies of the Spirurina, only four, the Gnathostomatoidea Railliet, 1895, Habronematoidea Chitwood & Wehr, 1932, Physalopteroidea Railliet, 1893 and Thelazioidea Skryabin, 1915, contain species that are parasitic as adults in freshwater, brackish-water or marine fishes, whereas members of the remaining six spirurine superfamilies are parasites exclusively of amphibians, reptiles, birds and mammals [5,6]. Each of these superfamilies with fish parasites comprises a single family in which these fish nematodes are placed: Cystidicolidae Skryabin, 1946, Gnathostomatidae Railliet, 1895, Physalopteridae Railliet, 1893, and Rhabdochonidae Travassos, Artigas & Pereira, 1928 [12].
To date, the fauna of spirurine nematodes, as well as of other nematodes parasitizing marine fishes in New Caledonian waters, has not been well characterized. The spirurine family Gnathostomatidae is so far represented here by two species of Echinocephalus Molin, 1858, both parasites of elasmobranchs (rays) [17], whereas representatives of five genera belonging to the Cystidicolidae, i.e. Ascarophis van Beneden, 1871 ( and Spinitectus Fourment, 1884 (1 species) have been reported from teleosts off New Caledonia and the nearby Chesterfield Islands [18][19][20][21]. The family Rhabdochonidae is represented here by a single species of Johnstonmawsonia Campana-Rouget, 1955 [15]. However, no species of the Physalopteridae has hitherto been recorded from New Caledonian fishes.
In 2009 and 2011, during extensive studies of the parasites of marine fishes in New Caledonian waters, physalopterid nematodes referable to the genus Bulbocephalus Rasheed, 1966 were collected from the digestive tract of the Cinnabar goatfish Parupeneus heptacanthus (Lacépède), the Indian goatfish Parupeneus indicus (Shaw) (both Mullidae, Perciformes), and the fish (no common name) Dentex fourmanoiri Akazaki & Séret (Sparidae, Perciformes). Closer examination of these nematodes revealed that they represent two new species. Results of the evaluation of these specimens are presented herein.
Whereas P. indicus and P. heptacanthus are tropical, reefassociated commercial fishes widespread in the Indo-Pacific region, D. fourmanoiri is a rare, deep-water fish with a limited distribution in the Southwest Pacific, occurring near the Chesterfield Islands and New Caledonia [9].

Materials and methods
Fish were caught off New Caledonia by various means. The nematodes for morphological studies were fixed in hot 4% formalin or hot water then 70% ethanol. For light microscope examination (LM), they were cleared with glycerine. Drawings were made with the aid of a Zeiss microscope drawing attachment. Specimens used for scanning electron microscope examination (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 Fish-Base [9].  Watson, 1916) Amended diagnosis: Body filiform. Cuticle slightly transversely striated. Anterior end of body with 2 lateral triangular pseudolabia, each provided with distinct terminal tooth, 2 cephalic papillae located dorso-and ventrolaterally, and lateral amphid. Oral aperture oval, dorsoventrally elongate, with smooth margin. Oesophagus divided into short anterior muscular and long posterior glandular regions. Muscular oesophagus consists of 3 portions: anterior, expanded portion modified to form protrusible pouch-like organ with 2-4 lobes; middle portion enclosed, along with nerve ring, by thin muscular sac-like structure; and narrow posterior portion. Excretory pore posterior to nerve ring. Deirids small, of irregular shape, with 2-5 prongs on distal end. Male with ventral, precloacal area rugosa formed by longitudinal ridges. Spicules unequal and dissimilar; right spicule shorter. Male posterior end with subventral caudal alae. Four pairs of preanal and 6 pairs of postanal papillae. Uterus of female amphidelphic. Vagina directed anteriorly from vulva. Fully developed eggs thick-walled, oval, containing larvae; eggs non-filamented. Type species: R. inglisi (Rasheed, 1966).
Male (6 specimens from P. heptacanthus, with measurements of holotype in parentheses; measurements of 1 specimen     representing 37% of body length; anterior, protrusible part of muscular oesophagus 99 long and 87 wide; middle portion of muscular oesophagus 135 long, maximum width 72; posterior portion 81 long, width 60; glandular oesophagus 2.11 mm long and 144 wide; muscular sac-like structure enclosing middle part of muscular oesophagus and nerve ring 135 long and 120 wide. Deirids, nerve ring and excretory pore 180, 231 and 285 from anterior extremity, respectively. Vulva situated 4.39 mm from anterior end of body, at 67% of body length; vulval lips slightly elevated ( Figure 1G). Vagina muscular, directed anteriorly from vulva and short ovijector. Uteri containing numerous oval, thick-shelled, embryonated (larvated) eggs; eggs 36-39 · 27-30, with wall 3 thick. Eggs located in uterus near vulva covered by additional, light-coloured thick layer, appearing as kind of capsule with enclosed proper egg; size of eggs including such capsule 48-54 · 36-39. Tail very short, 21, with rounded tip (Figures. 1L, 4C).
From marine fishes of Pakistan, Rasheed [24] inadequately described two species of interesting spiruroid nematodes with protrusible anterior oesophageal lobes, for which she established the new monotypic genera Bulbocephalus (type species B. inglisi) and Cestocephalus Rasheed, 1966 (type species C. serratus Rasheed, 1966). Subsequently, based on similar nematodes collected from fishes in South Vietnam, Le-Van-Hoa, Pham-Ngoc-Khue & Nguyen-Thi-Lien [11] synonymized Cestocephalus with Bulbocephalus, which was followed by Chabaud [5,6] and subsequent authors; they also designated C. serratus of Rasheed [24], inadequately described from a single female, to be a species inquirenda. However, in accordance with the international rules, the genus is objectively determined by its type species and, consequently, Cestocephalus becomes a genus inquirendum and cannot be considered a synonym of Bulbocephalus (= Rasheedia). Therefore, the Vietnamese species similar to C. serratus should be tentatively reported as Cestocephalus petterae (Le-Van-Hoa, Pham-Ngoc-Khue & Nguyen-Thi-Lien, 1972) n. comb. Since the cephalic structures of both C. serratus and C. petterae are insufficiently studied, it is reasonable to consider Rasheedia and Cestocephalus [genus inquirendum] as possibly different genera. In contrast to Rasheedia spp., both representatives of Cestocephalus are allegedly characterized by the presence of three large oesophageal lobes resembling bothridia of cestodes, which form a hood or umbrella-like structure on the cephalic end, and by a deeply transversely striated cuticle appearing from the side to have sharp edges or serrations.
The original description of R. inglisi is inadequate, especially regarding the cephalic structures, and, as mentioned by Le-Van-Hoa et al. [11], the presence of pseudolabia was evidently overlooked by Rasheed [24]. Moreover, considering that the specimens were collected from two different species of hosts, it is not certain that the available nematode males and females belonged to one and the same species. According to Le-Van-Hoa et al. [11], R. deblocki is very similar to R. inglisi, differing from it only in the presence of a single point (instead of allegedly three small points) on the female tail tip, ventral precloacal cuticular ridges in the male and in somewhat greater measurements. However, it is highly probable that Rasheed [24] considered a pair of phasmids, situated near the female tail tip as illustrated for R. deblocki, to be terminal points and she apparently overlooked the presence of precloacal ridges in R. inglisi. Since both of these nematode species were collected from E. tetradactylum in the Indo-West Pacific region, it is highly probable that subsequent studies will prove their conspecificity; some biometrical differences may well be within the intraspecific variability of a single species. However, for the time being and until new material is available, we consider R. inglisi and R. deblocki to be separate species.
Rasheedia heptacanthi n. sp. is easily distinguished from R. inglisi and R. deblocki by distinctly smaller body measurements, the longer left spicule and the unusual structure of eggs. It differs from R. pseudupenei mainly in having longer spicules, different structure of eggs and in the geographical  Prevalence and intensity: 1 fish infected/5 fish examined; 2 specimens.
Deposition of type specimen: Helminthological Collection, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Č eské Budějovice, Czech Republic (male holotype mounted on SEM stub, Cat. No. N-1162). Larval specimen not deposited, intended for sequencing.
Etymology: The scientific name novaecaledoniensis relates to the country, i.e. New Caledonia, near the coast of which the fish host of this nematode was collected.

Remarks
This new species differs from other congeners in possessing four (instead of two or three) anterior protrusible oesophageal lobes. In addition, R. inglisi and R. deblocki have much larger body measurements and they are parasites of the Polynemidae (vs. Mullidae). In contrast to R. heptacanthi n. sp., the muscular oesophagus in the portion anterior to the nerve ring of R. novaecaledoniensis n. sp. is not expanded and the spicules are distinctly shorter.
Deirids of R. pseudupenei are located much more anteriorly to the level of the nerve ring as compared with those in R. novaecaledoniensis n. sp. and both species differ in the genus of hosts and in distant geographical regions (North Atlantic vs. South Pacific) (see the key to species of Rasheedia at the end of Discussion).
From the intestine of the same host species (P. indicus) in the Indian Ocean off Somalia, another spirurine nematode, Ascarophis parupenei Moravec, Orecchia & Paggi, 1988 (Cystidicolidae, Habronematoidea) was described [23]. However, the cephalic structure of this nematode is very different from that of R. novaecaledoniensis n. sp. and the spicules are much longer (533-600 lm and 150-171 lm vs. 381 lm and 87 lm).

Discussion
The present study shows that the morphology of Rasheedia spp. is very unusual among parasitic nematodes, namely in possessing the anterior portion of the muscular oesophagus F. Moravec and J.-L. Justine: Parasite 2018, 25, 39 modified to form a protrusible pouch-like organ with anterior lobes and the middle portion of the muscular oesophagus enclosed in a thin-walled muscular sac-like structure. These features are already present in the third-stage larva of R. heptacanthi examined (Figures 2A, 2B). All previously described species of Rasheedia (reported as Bulbocephalus) were studied only by LM and probably due to the very limited numbers of available specimens, and the fact that some morphological features are not easily visible using LM, the existing species descriptions are evidently incomplete and may even be somewhat misleading.
The present study of two species of Rasheedia by SEM, used for the first time for nematodes of this genus, made it possible to describe in detail the cephalic structures. Whereas the presence of three protrusible oesophageal lobes was reported for all the three previously described species of Rasheedia, the SEM micrographs revealed two or four lobes in the two newly established species. Although Rasheed [24] did not mention the presence of deirids in R. inglisi, these were reported (but not illustrated) for R. deblocki [11] and for R. pseudupenei [25], but their shape was not described. The SEM micrographs of R. heptacanthi n. sp. showed considerable intraspecific variability in the shape of deirids in adults of this species, which may be from bifurcate ( Figures 2I, 5H) to possessing up to five distal prongs ( Figures 2G, 2H, 2J, 5F, 5G). The shape of deirids is usually stable within the same species in spirurids and is frequently considered to be a reliable interspecific taxonomic feature in some groups, for example in species of Rhabdochona Railliet, 1916 (Rhabdochonidae, Thelazioidea) [13]. However, in contrast to adults of R. heptacanthi n. sp., the conspecific third-stage larva was found to possess simple, rounded deirids ( Figures 2D, 5E). The presence of a small ventral median caudal protuberance, found in the males of both new species of Rasheedia, was not previously reported for representatives of this genus. Such a caudal protuberance has so far been observed only in some cystidicolid nematodes [8,15,19,22].
A remarkable feature of R. heptacanthi n. sp. is that the fully developed eggs located in the uterus near the vulva have an additional thick outer layer, appearing like a capsule enclosing the true egg ( Figure 2F). As far as the authors know, among fish nematodes, mature eggs covered by an additional thick outer layer have only been described in a few species of capillariids (Capillariidae) parasitizing marine fishes, for example Gessyella latridopsis (Johnston & Mawson, 1945) or Capillaria appendigera Moravec & Barton, 2018 from Australian waters [10,14], but these are not known in species of the Spirurida.
The third-stage larva of R. heptacanthi n. sp. is the first infective larva of Rasheedia described so far. Its morphology is very similar to that of conspecific adults, but its deirids are simple and the tail tip is provided with a cuticular knob, resembling thus third-stage larvae of many other spiruride nematodes parasitizing fishes as adults. It can be assumed that, as in most spirurides parasitizing marine fishes, the life cycles of Rasheedia spp. involve arthropods (probably crustaceans) as intermediate hosts. Bilqees [3]. However, the illustrations of the cephalic end of P. macrolabiata are reminiscent of a species of Rasheedia and this possibility is also supported by the type of the host (marine fish). Unfortunately, both the description and illustrations of these nematodes are poor and confusing, making it necessary to consider Pseudomazzia Bilqees, Ghazi & Haseeb, 2005 and P. macrolabiata to be nomina dubia.