Bucephalus damriyasai n. sp. (Digenea: Bucephalidae) from the blacktip trevally Caranx heberi (Bennett) (Perciformes: Carangidae) off Bali, Indonesia

The new species Bucephalus damriyasai n. sp. is described from Caranx heberi (Bennett) from off Bali, Indonesia. It can be distinguished from other Bucephalus spp. recorded from carangid hosts by its narrow elongate body shape and the relatively long distance between the rhynchus and the vitellarium, as well as other features distinguishing it from individual species. The most similar species are differentiated from B. damriyasai n. sp. as follows: B. carangis Yamaguti, 1970 has a much greater length, the rhynchus is smaller and the cirrus-sac is small, not always reaching to the posterior testis; B. fragilis Velasquez, 1959 is a tiny species, the pre-vitelline distance is short and the caecum is saccular; B. gorgon (Linton, 1905) is much longer and relatively broader, the uterus reaches distinctly anterior to the vitellarium and the rhyncheal tentacles appear more complex; B. labracis Paggi & Orecchia, 1965 is distinctly longer, slightly broader, with a slightly larger rhynchus, and has shorter pre-uterine and pre-mouth distances; B. paraheterotentaculatus Velasquez, 1959 is much longer, relatively rather broad, the rhynchus is said to bear 21 tentacles, the post-testicular region and cirrus-sac reach are longer and the caecum is described as saccular; B. sphyraenae Yamaguti, 1952 is longer, slightly broader, the uterus reaches anteriorly to the vitellarium and the caecum is claviform and oriented anteriorly; B. margaritae Ozaki & Ishibashi, 1934 (syn. B. varicus Manter, 1940) is relatively squat, has shorter pre-vitelline and pre-mouth distances and a longer post-testicular distance and cirrus-sac reach; B. yamagutii Gupta & Singh, 1985 is broader, with a relatively short pre-vitelline distance, the caecum extends anteriorly to the pharynx, but not posteriorly and the rhynchus is said to carry five tentacles. The distinctive features of B. damriyasai n. sp. are compared with those of all other marine Bucephalus spp. in a table. The number of bucephalid trematodes known from Indonesian waters is now 13, two of them await further identification. They have been described from the fish families Carangidae, Platycephalidae, Sciaenidae, Serranidae and Sphyraenidae.


Introduction
The Indonesian fish parasite fauna is species-rich, resulting from the high number of fish species surrounding about 17,000 islands of this maritime nation. However, though being a hot spot of aquatic biodiversity, fish parasites are far less studied. As stated by Bray & Palm (2009) and considering recent studies Yong et al., 2016;Bray et al., 2017), over 80 fish-parasitic trematode species have been reported from Indonesian waters.
According to Palm & Bray (2014), many fish parasites that have been reported off the Hawaiian Islands in the Central Pacific have an Indo-Pacific or even worldwide distribution. Although it can be expected that a high number of new trematode taxa will be described from Indonesia, the same importance should be placed on having reliable identifications of already known species from other regions. Without proper identification the use of fish parasites as biological indicators (Palm, 2011), of increasing importance in many regions (Truong et al., 2017), is difficult and in some cases impossible. , Palm et al. (2011) and Neubert et al. (2016) have developed a method to use grouper fish parasites as biological indicators for pollution and environmental change in Indonesian coastal waters, but several bucephalids recovered were only tentatively identified (Bray & Palm, 2009).
The genus Bucephalus Baer, 1826 includes many species reported from fresh and marine waters. It is characterised by having a sucker-like rhynchus with a hood bearing tentacles (usually seven). The tentacles may be found withdrawn and difficult to see and in such cases the worms look very similar to members of Rhipidocotyle Diesing, 1858. Recent molecular studies by Nolan et al. (2015) have indicated that the genus is polyphyletic, with at least three separate monophyletic groups embedded within an assortment of species of Rhipidocotyle, Prosorhynchoides Dollfus, 1929 andPaurorhynchus Dickerman, 1954. Caranx heberi (Bennett) (syn. C. sem Cuvier) is a common carangid throughout the Indo-Pacific region. The only reports of digeneans we are aware of from this host are the bucephalid Bucephalus margaritae Ozaki & Ishibashi 1934 from off Natal, South Africa (Bray, 1984) and an unidentified sclerodistomid Prosorchis sp. from the Arabian Gulf (El-Naffar et al., 1992;Al Kawari et al., 1996). We herewith present a description of new bucephalid species from this host from the Balinese coast, Indonesia.

Materials and methods
The present study is based on material collected during the First Educational Workshop on Marine Fish Parasites in Bali, July 21st -August 2nd 2013, of Indonesian and international students and researchers investigating a wide range of hosts from Balinese waters. Three specimens (15.7-16.7 cm total length, 66.2-70.6 g) of Caranx heberi were caught by artisanal fishermen and landed at Kedonganan Bay, transported alive into the laboratory of the Veterinary Faculty, Udayana (UNUD) University, Denpasar, Bali, and directly studied for fish parasites. Kedonganan Bay is located at the western side of the southern tip of Bali, directly next to the Ngurah Rai Kuta international airport. The airstrip of the airport reaches into the ocean and acts as the northern border of the bay. Kedonganan is a typical fishing village but is heavily influenced by tourism. There is no harbour; the small ships lay directly in front of the beach which is used to land captures. The fishermen catch fish from the Bali Strait and from nearby areas off South Bali and East Java. They use drift nets, troll lines and hand lines. A cooperation of the local fishermen manages the market (Proctor et al., 2003). Digeneans were collected according to the gut wash methodology described by Cribb & Bray (2010). Unfortunately, although these worms were collected with a view to sequencing, these specimens have not yielded usable rDNA.
Whole-mounts were stained with Mayer's paracarmine, cleared in beechwood creosote and mounted in Canada balsam. Measurements were made through a drawing tube on an Olympus BH-2 microscope, using a Digicad Plus digitising tablet and Carl Zeiss KS100 software adapted by Imaging Associates, and are quoted in micrometres, as the range and the mean in parentheses. 'Cirrus-sac reach' is the distance between the posterior extremity of the worm and the anteriormost extent of the cirrus-sac. The type-material is deposited in the following museum collections: the Natural History Museum, London, UK (NHMUK); the National Biodiversity Collection, Museum Zoologicum Bogoriense, Cibinong, Bogor, Java, Indonesia (MZB); and the Natural History Museum, Berlin, Germany (ZMB).
Excretory pore terminal; anterior extent of vesicle visible in some specimens reaching just anterior to vitellarium.

Discussion
We are aware of 48 described nominal species of Bucephalus in marine fishes and we have examined the descriptions of all except B. arabiana Varma, 1982, the description of which we have been unable to find. We have developed a visual key similar to that to Prosorhynchus developed by Bray & Palm (2009) (http://www.nhm.ac.uk/bray2009). Ten characters are used, most of which are listed as a percentage of body length: 1, Length; 2, Width %; 3, Rhynchus length %; 4, Tentacle number; 5, Pre-vitelline distance %; 6, Preuterine distance %; 7, Pre-mouth distance %; 8, Posttesticular distance %; 9, Cirrus-sac reach %; and 10, Egg length.
Bucephalus carangis Yamaguti, 1970 is reported from the black jack Caranx lugubris Poey and the bluefin trevally C. melampygus Cuvier off Hawaii (Yamaguti, 1970;Palm & Bray, 2014). It apparently grows to a much greater size than B. damriyasai n. sp. (to 3,500 lm), the rhynchus is smaller (3-5% of body length), and the cirrus-sac is small, not always reaching to the posterior testis (cirrus-sac reach about 18% of body length).
Bucephalus gorgon (Linton, 1905) (syn. Bucephalus introversus Manter, 1940) is a widely reported species known only from carangids and mainly from members of the genus Seriola in the Pacific, Indian and Atlantic Oceans. It was described by Linton (1905) as Gasterostomum gorgon in the yellowtail amberjack Seriola lalandi Valenciennes from the North-West Atlantic at Beaufort, North Carolina. Eckmann (1932) placed this species in Bucephalus. It was redescribed and illustrated (apparently a badly contracted specimen) by Linton (1940) as Nannoenterum gorgon from S. lalandi at Woods Hole, Massachusetts. Bartoli et al. (2005) redescribed this species from the greater amberjack Seriola dumerili (Risso) off Corsica in the western Mediterranean Sea. They considered Bucephalus introversus Manter, 1940, from the crevalle jack Caranx hippos (L.), S. dumerili, S. lalandi and Seriola sp. from the eastern Pacific off Mexico and Columbia (Manter, 1940a) as a synonym of B. gorgon. Further descriptions and descriptive matter have been given by Oshmarin (1965) from Seriola 'nigromaculata' off Vietnam; Corkum (1967) -Bernal, 1956;Parukhin, 1966;Nahhas & Powell, 1971;Parukhin, 1976;Fischthal, 1982;Montero et al., 2002;Gijon-Botella et al., 2007;Hutson et al., 2007). Nolan et al. (2015) used sequences of B. gorgon from S. dumerili in the Gulf of Mexico to show that the closest sequenced relative is Prosorhynchoides ovatus (Linton, 1898). Linton (1905, p. 364) described the ''anterior sucker (i.e. a rhynchus) surrounded by a crown of about eighteen tentacles''; in most cases, the anterior end of his specimens was withdrawn (his figure 241; incorrectly oriented). Linton (1940) redescribed the species with an anterior sucker provided with about 20 tentacles, the specimens being ''all macerated''. Bartoli et al. (2005) stated that ''the rhynchus of B. gorgon consists of seven large retractile tentacles, each of them provided with one or two small basal processes''. Bucephalus gorgon is usually described as much longer and relatively broader than B. damriyasai n. sp., the uterus reaches distinctly anterior to the vitellarium. The rhyncheal tentacles appear much more complex in B. gorgon, particularly as described by Bartoli et al. (2005).
Bucephalus paraheterotentaculatus Velasquez, 1959 was originally reported in the blackbanded trevally Seriolina [as Seriola] nigrofasciata (Rüppell) from Malabon, Rizai, Luzon Island, Philippines (Velasquez, 1959). It has subsequently been reported in S. nigrofasciata, S. dumerili and Seriola sp. from the South China Sea and Masirah Bay off Oman in the Northern Indian Ocean (Parukhin, 1966(Parukhin, , 1976. This species grows to 4,070 lm long, is relatively rather broad (maximum width about 12-13% of length), the rhynchus bears ''21 tentacles grouped in multiples of 3 conforming to the basic number of 7'', the posttesticular region is about 31% of body length and the cirrus-sac reach is about 36% of body length. The caecum is described as saccular.
Bucephalus varicus Manter, 1940 is usually considered one of several synonyms of B. margaritae Ozaki & Ishibashi, 1934. A fairly high proportion of records of Bucephalus species from carangids are of B. margaritae and its synonyms. It was originally described as a furcocercous cercaria from the pearl oyster Pinctada imbricata Röding (as Pinctada imbricata mertensii) off Japan (Ozaki & Ishibashi, 1934). A series of papers by Sakaguchi (1962Sakaguchi ( , 1964Sakaguchi ( , 1966aSakaguchi ( , b, 1968) reported on the completion of the life-cycle of this species and concluded that it was conspecific with B. varicus Manter, 1940. Manter (1940a in describing B. varicus from ''a young specimen of an unidentified species of Caranx, or jack'' off Bahia Honda, Panama, considered that B. polymorphus of Nagaty (1937) from carangids in the Red Sea was a misidentification, as B. polymorphus is a freshwater species. Overstreet (1969) and Velasquez (1975) considered B. pseudovaricus Velasquez, 1959synonymous with B. varicus. Bray (1984 also considered that B. retractilis Yamaguti, 1959, B. carangoides Yamaguti, 1970and B. ulua Yamaguti, 1970 are synonyms of B. margaritae. Nahhas et al. (2006) ''confirm this synonymy'' and described the worm from the cleftbelly trevally Atropus atropos (Bloch & Schneider), the largemouth queenfish Scomberoides commersonnianus Lacépède, the Malabar trevally Carangoides malabaricus (Bloch & Schneider), the whipfin silver-biddy Gerres filamentosus Cuvier and the pickhandle barracuda Sphyraena jello Cuvier in the Arabian Gulf off Kuwait. Chinchilla et al. (2006) accepted these synonymies, and described the worm from the southern sennet Sphyraena picudilla Poey off Venezuela. Marchiori et al. (2010) also accepted the synonymy and described the life-cycle in the brown or South American rock mussel Perna perna (L.), the combtooth blenny Hypleurochilus fissicornis (Quoy & Gaimard) and the southern kingcroaker Menticirrhus americanus (L.) in the waters off Brazil. Many of the more recent records of B. margaritae are of non-carangid hosts. Al-Zubaidy (2011) described B. margaritae and B. varicus as separate species, from the great barracuda Sphyraena barracuda (Edwards) and the orange-spotted trevally Carangoides bajad (Forsskål), respectively, from Yemeni Red Sea coastal waters off Hodeidah. The illustrations suggest that several species are involved. Nolan et al. (2015) used sequences of worms identified as B. margaritae from Caranx crysos (Mitchill), from the Gulf of Mexico in their molecular study and showed that, of sequenced species, it is the sister of B. cynoscion Hopkins, 1956. Bucephalus margaritae differs from B. damriyasai n. sp. in its relatively squat shape, short pre-vitelline distance, shorter pre-mouth distance and longer posttesticular distance and cirrus-sac reach. The species has been described many times under the same or different names and clearly needs careful revision. It is likely, if not virtually certain, that a complex of similar species is now known under this name.
Bucephalus yamagutii Gupta & Singh, 1985 is reported only from the Malabar trevally Carangoides malabaricus (Bloch & Schneider) (as Caranx malabaricus) off the Puri coast in the Bay of Bengal (Gupta & Singh, 1985). It is relatively broad (width about 16% of length), with a relatively short previtelline distance (about 28% of body-length) and the caecum extends anteriorly to the pharynx, but not posteriorly. The rhynchus is said to carry five tentacles.
The features that distinguish marine Bucephalus spp. from B. damriyasai n. sp. are tabulated in Table 1.

Concluding remarks
It is not clear why we have not been successful in securing useful DNA from this species as it was fixed in the same way as other digeneans recovered from the Bali Workshop that have been successfully sequenced (Cribb et al., 2014;Bray et al., 2016;Yong et al., 2016;Bray et al., 2017). Successful and experienced molecular biologists in the Rostock and the University of Queensland Laboratories have been frustrated in their attempts to extract DNA from these worms. The species described here is morphologically distinct enough to be easily recognised so it was felt worthwhile to describe it and add a further detail to our depauperate knowledge of the marine fish digeneans of Indonesia.