© Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2016, 63: 026 doi: 10.14411/fp.2016.026 http://folia.paru.cas.cz Research Article A morphological and genetic description of pentastomid infective nymphs belonging to the family Sebekidae Sambon, 1922 in fish in Australian waters Diane P. Barton1,2,3 and Jess A.T. Morgan4,5 1 Fisheries Research, Department of Primary Industries & Fisheries, Berrimah Farm, Darwin, Northern Territory, Australia; 2 Research Institute for the Environment and Livelihoods, Charles Darwin University, Darwin, Northern Territory, Australia; 3 Museum and Art Gallery of the Northern Territory, Conacher Street, Fannie Bay, Darwin, Northern Territory, Australia; 4 Queensland Alliance for Agriculture and Food Innovation, Centre for Animal Science, The University of Queensland, St. Lucia, Queensland, Australia; 5 Animal Science, Queensland Department of Agriculture and Fisheries, EcoSciences Precinct, Dutton Park, Queensland, Australia Abstract: Infective nymphal stages of the family Sebekidae Sambon, 1922 are reported from four species of fish in Australian waters for the first time. Infected fish were collected from locations in Western Australia, the Northern Territory and north Queensland. The in- fective nymphs of Alofia merki Giglioli in Sambon, 1922 and Sebekia purdieae Riley, Spratt et Winch, 1990 are reported and described for the first time. The remaining specimens were identified as belonging to the genusSebekia Sambon, 1922 based on the combination of buccal cadre shape, shape and size of hooks, and overall body size, but could not be attributed to any of the other species of Sebekia already reported due to missing required morphological features. DNA sequences of members of the family Sebekidae are presented for the first time. The lack of knowledge on the pentastome fauna of wild crocodiles, and any potential intermediate hosts, in northern Australia, is also outlined. Keywords: Protonibea, Lutjanus, Lates, Ambassis, intermediate host, DNA This article contains supporting information (Tables S1–S6) online at http://folia.paru.cas.cz/suppl/2016-63-026.pdf During a survey of the parasite fauna of various fish Most larval sebekid pentastomes reported from fish ma- species in northern Australian waters, pentastomid infec- ture in crocodiles (Junker et al. 1998, Guidelli et al. 2003, tive nymphs, identified as belonging to the family Sebeki- Almeida et al. 2010, Giesen et al. 2013), with a few species dae Sambon, 1922, were collected. Although commonly maturing in freshwater turtles (Curran et al. 2014). Croco- reported within fish hosts elsewhere (Junker et al. 1998, diles in the Northern Territory, Australia, have been report- Guidelli et al. 2003, Almeida et al. 2010, Giesen et al. ed with at least five species belonging to three genera, all 2013), no larval pentastomids have previously been report- Sebekidae, from both wild and farmed populations (Riley ed from fish in Australian waters. et al. 1985, 1990, Riley 1994, Riley and Hurchzermeyer Descriptions of infective nymphal stages of many of 1996). At present, however, nymphs (also collected from the sebekid species are not available, making identification crocodiles) have only been described for two of those spe- of infections from intermediate hosts difficult. However, cies (Riley et al. 1990). Winch and Riley (1986) suggested that the infective larvae Although the distribution of crocodiles ranges across the of Sebekia oxycephala (Diesing, 1836) (collected from fish) whole of northern Australia (Webb and Manolis 1989), in- possessed the final number of annuli as for the adult stage. cluding areas of Western Australia and Queensland, no pen- Infective nymphs were identified as those in possession of tastomes have been reported from crocodiles outside of the ‘double hooks’ (i.e. hooks with the dorsal accessory piece) Northern Territory. Little is known about the ecology of in- and well defined annuli fringed by a single row of spines fection of pentastomids in wild crocodiles in northern Aus- (Winch and Riley 1986). If this is true for all sebekids, the tralia or of their distribution in potential intermediate hosts. nymphal stages collected in this study were infective and This paper presents the first record of nymphal pentas- could potentially be identified to species by annulus count. tomes from fish in Australian waters and also provides the Address for correspondence: D.P. Barton, Fisheries Research, Department of Primary Industries & Fisheries, Berrimah Farm, Darwin, Northern Terri- tory 0801, Australia. Email: [email protected] This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2016.026 Barton and Morgan: Pentastomid nymphs in Australian fish Table 1. Locations of fish collected from along the coastlines of Western Australia (WA), the Northern Territory (NT) and Queensland (QLD) examined in the present study. Locations are listed in order travelling from west to east. Number of fish examined from each location is presented, with the range in mm, mean of total length (TL) in parentheses. Protonibea diacanthus (Lacepède) Lutjanus johnii (Bloch) Lethrinus laticaudis Alleyne et Macleay State Location No. Mean TL No. Mean TL No. Mean TL Pilbara - - - - 69 259–492 (363) Broome 36 720–1 199 (1 019) - - - - WA Kimberleys 20 520–920 (647) 61 240–689 (379) 101 220–477 (333) Wyndham 34* 804–1 220 (1 067) - - - - Bonaparte Gulf - - 23 447–569 (517) - - Wadeye 25 540–1 160 (789) 27 415–560 (463) 30 290–440 (396) Bynoe Harbour 25 N/A 26 250–545 (352) 11 220–480 (341) Darwin Harbour 26 395–1 150 (654) 75 150–710 (344) 56 175–395 (257) Cape Hotham 10* 430–1 230 (990) 9 345–425 (383) - - Bathurst Island 28* 387–1 235 (969)† 36 247–540 (379) - - NT Melville Island 30 405–1 170 (646) 31 240–595 (339) - - Coburg Peninsula - - 35 322–631 (461) 33 230–370 (309) Maningrida 29* 420–1 210 (731) 16 270–510 (368) - - Arnhem Land 19 N/A 66 200–625 (411) - - Gove 1 360 - - - - Groote Eylandt/Blue Mud Bay 3 580–850 (760) 58 370–770 (523) 29 260–380 (307) Borroloola 29 440–740 (588) 25 310–645 (407) 30 245–433 (308) Gulf of Carpentaria - - 21 355–710 (537) 3 323–343 (333) QLD Halifax Bay - - 51* 364–822 (596) 14 239–405 (354) South East Qld - - - - 37 268–547 (383) Total 315 560 413 * infected fish – data regarding these locations are presented in the text; N/A – fish where TL data was not obtained; † fish collected from Aspley Strait not included in TL data presented. first DNA sequences of members of the Sebekidae which August 2015. Nine P. diacanthus were collected from the Aspley will aid in future diagnostics of the group. Strait (11°29'57S''; 130°24'32''E) on 10 September 2015. A total of 29 P. diacanthus were collected from Maningrida in August 2014 MATERIALS AND METHODS and July 2015. Two sites were sampled near Maningrida: Liv- Fish from the following species were examined for parasites erpool River and Blythe River. Twenty-four P. diacanthus were as elements of a larger project: the black jewfish, Protonibea di- collected from the Liverpool River (12°04'16''S; 134°11'21''E) on acanthus (Lacepède) (Sciaenidae), the golden snapper, Lutjanus 25–27 August 2014. Five P. diacanthus were collected from the johnii (Bloch) (Lutjanidae) and the grass emperor, Lethrinus lat- Blythe River (12°05'38''S; 134°36'09'') on 28 July 2015. icaudis Alleyne et Macleay (Lethrinidae). Of these species, fish Infected L. johnii were collected from Halifax Bay, north of belonging to P. diacanthus and L. johnii were found to be infected Townsville, northern Queensland (18°32'14''S; 146°21'11''E). with pentastomid nymphs (Table 1). A total of 51 L. johnii were collected from May to December Infected P. diacanthus were collected from four separate lo- 2014. cations: Wyndham (Western Australia), Cape Hotham, Bathurst All fish were euthanased (Charles Darwin University Animal Island and Maningrida (the Northern Territory). A total of 34 Ethics Approval A13014), placed on ice and transported to the P. diacanthus were collected from Cambridge Gulf, near Wyn- laboratory for processing; some fish were frozen whole prior to dham (14°49'22''S; 128°17'15''E) in May and June 2015. A total processing. All internal organs were removed for examination for of 10 P. diacanthus were collected from Cape Hotham in August parasites. The intestinal tract, and associated mesenteries, were 2013 and April and May 2014. Two sites were sampled at Cape separated, placed in saline and examined under a dissector mi- Hotham: Ruby Island off the eastern tip and the mouth of the croscope for the presence of parasites. Pentastome nymphs were Adelaide River at the western base. Four P. diacanthus were col- found encysted in the mesenteries along the intestinal system. lected from Ruby Island (12°06'28''S; 131°20'59''E) on 30 August The nymphs were removed from their cysts and preserved in 70% 2013. Six P. diacanthus were collected from the mouth of the Ad- ethanol. elaide River (12°13'12''S; 131°14'1.5''E) between 25 April and 11 Nymphs were cleared using lactophenol solution and observed May 2014. Four sites were sampled around Bathurst Island: Port intact or partially intact using light microscopy. Hooks and the Hurd (western side), Caution Point (western side), St Asaph Bay oral cadre were dissected from some specimens. Hook dimen- (northern side), and Aspley Straits (running between Bathurst and sions measured were blade length (AC), hook length (AD), base Melville Islands). Three P. diacanthus were collected from Port length (BC), plateau length (CD), and hook gape (AB) (Fig. 1). Hurd (11°38'52''S; 130°12'36''E) on 11 November 2013.