Parasitology International 64 (2015) 397–404 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Characterisation of Ascaridoid larvae from marine fish off New Caledonia, with description of new Hysterothylacium larval types XIII and XIV Shokoofeh Shamsi a,⁎, Anita Poupa a,Jean-LouJustineb a School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, Australia b ISYEB, Institut de Systématique, Évolution, Biodiversité (UMR7205 CNRS, EPHE, MNHN, UPMC), Muséum National d'Histoire Naturelle, CP 51, 55 rue Buffon, 75231 Paris cedex 05, France article info abstract Article history: Here we report occurrence of six different morphotypes of ascaridoid type larvae from 28 species of fish collected Received 17 November 2014 from New Caledonian waters. The larvae were morphologically identified as Anisakis type I, Hysterothylacium Received in revised form 12 May 2015 type VI and new larval types XIII and XIV, Raphidascaris larval type and Terranova larval type II. Representatives Accepted 16 May 2015 of each morphotype were subjected to the amplification of the second internal transcribed spacers (ITS-2) of ri- Available online 23 May 2015 bosomal DNA (rDNA) and those sequences were compared with ITS-2 sequences of other ascaridoid nematodes Keywords: previously deposited in GenBank. ITS-2 sequences of Anisakis larval type I were identical to those of A. typica. ITS- Anisakidae 2 sequences of Hysterothylacium larval type VI in the present study were identical to those previously found in Raphidascarididae Eastern Australian waters. No match was found for ITS-2 sequences of Hysterothylacium larval types XIII and Fish XIV; therefore, the specific identities of these larval types remain unclear. ITS-2 sequences of Raphidascaris larval Parasites type were identical to those of R. trichiuri, which have previously been reported in Taiwanese waters. Terranova New Caledonia larval type II in the present study had identical ITS-2 sequences with Terranova larval types reported from Australian waters, however, the specific identity is unknown. This taxonomic work is essential if further research on these zoonotic parasites is to be effective. This includes investigations into such aspects as life cycle studies, impacts on human health and risk assessment for their transmission to humans. © 2015 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Caledonia in order to elucidate the taxonomy of these important and potentially zoonotic parasites in that region. Ascaridoid nematodes have a worldwide distribution [1]. The life cycle of aquatic ascaridoids usually involves a predatory fish, marine 2. Materials and methods mammal or piscivorous bird as the definitive host and a broad range of aquatic invertebrates and fish species as the intermediate/paratenic 2.1. Parasite collection host [1]. Marine ascaridoids, particularly anisakids, have attracted con- siderable attention following the discovery by Van Thiel [2] that the lar- The host fish were either fished off Récif Toombo, off Nouméa, New val stages of Anisakis from the North Sea herring, Clupea harengus, are Caledonia (coordinates: 22°35′S, 166°29′E), off Ile Redika, off Nouméa able to infect humans. Since then, there have been an increasing number (22°32′S, 166°37E), or obtained from the fish market of Nouméa City of publications revealing various aspects of their biology and ecology (Table 2), in 2009–2010. Fish from the market were obtained fresh [3]. However, in New Caledonia, little is known about these important from mackerel fishermen that fish in a 30 km radius around Nouméa. parasites. There are many publications reporting and describing para- The fish were routinely photographed, measured (fork length) and sites infecting marine fish in New Caledonian waters [4, 5], however weighed, and assigned a Muséum national d'Histoire naturelle only a few publications included the identification of ascaridoid nema- (MNHN) JNC number which was consecutively used for parasites. todes at the species or genus level (Table 1), and several of them report- Identification of the fishwasdonebyJLJ,andwasconfirmed by ich- ed the occurrence of ascaridoid nematode larvae in many fish species as thyologists. As often occurs in high biodiversity areas in the South Pacif- “anisakid larvae” without further molecular identification [6–8].There- ic, the systematics of certain fish were problematic. Carangids were fore, the aim of the present study is to morphologically describe and ge- especially dif ficult to identify. Certain specimens of Carangoides sp. netically characterise ascaridoid larvae from marine fish off New were similar in many aspects to the published description of C. talamparoides Bleeker, but with differences in colour that prevented definitive identification. Specimens of C. chrysophrys were similar to ⁎ Corresponding author. the published description but with minor differences in colour; one of E-mail address: [email protected] (S. Shamsi). the ichthyologists consulted did not exclude the possibility that it http://dx.doi.org/10.1016/j.parint.2015.05.014 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved. 398 S. Shamsi et al. / Parasitology International 64 (2015) 397–404 Table 1 Ascaridoid nematodes mentioned in marine fish off New Caledonia with a specific or generic identification. Anisakid larvae without further identification were mentioned in many fish species [6–8]. Ascaridoid nematode species Stage Fish host Reference Anisakis sp. Larvae Epinephelus areolatus [6] Anisakis sp. Larvae Nemipterus furcosus [8] Hysterothylacium cenoticum Adults Tetrapturus audax [5] Hysterothylacium sp. Larvae Plectropomus laevis [6] Hysterothylacium sp. Larvae Nemipterus furcosus [8] Raphidascaris (Ichthiascaris) nemipteri Adults Nemipterus furcosus [5] Raphidascaris (Ichthiascaris) etelidis Adults & juveniles Etelis coruscans [28] Pristipomoides filamentosus Raphidascaris (Ichthiascaris) sp. Juveniles Lutjanus vitta [28] Lethrinus genivittatus Lethrinus miniatus Lethrinus rubrioperculatus Terranova scoliodontis Adults Galeocerdo cuvier [29] Terranova sp. Larvae Epinephelus cyanopodus [30] Terranova sp. Larvae Epinephelus areolatus Variola albimarginata Variola louti Terranova sp. Larvae Lutjanus vitta [8] could be a new species. Parasitological results have already been stained with GelRed™and photographed using a gel documentation published about these Carangoides spp. [9]. The systematics of system. Gymnocranius (Lethrinidae) was recently re-assessed [10, 11]; the spec- imens we examined for parasites were simultaneously examined by 2.3.2. Sequencing ichthyologists and one was attributed to the recently erected species Representatives of each morphotype (see Section 2.2. above) was se- fi Gymnocranius superciliosus [11]. In some cases, the identi cation of lected for sequencing. Amplicons were purified over mini-columns fi fi sh used in this study was con rmed by molecular analyses of COI se- (Wizard™ PCR Prep, Promega,WI, USA), eluted in 35 μl H2O and then quences (barcoding). This was the case for specimen JNC3126 of subjected to automated sequencing using the same primers as for PCR. Selar crumenophtalmus [12] and specimen JNC3142 of Epinephelus Sequences were aligned using the computer programme ClustalX and fi chlorostigma [13].ForAlepes vari and Carangoides fulvoguttatus, identi - then adjusted manually. Polymorphic sites were designated using Inter- fi cation was con rmed by COI sequences of other specimens with similar national Union of Pure and Applied Chemistry (IUPAC). morphologies [9, 12]. The abdominal cavities of the fish were opened and parasites were 3. Results collected by the wash method [14]. Anisakids were found mainly from the intestinal lumen, but some came from encapsulations on the surface Table 2 shows a summary of infected fish and the list of parasites of the abdominal organs. These specimens were fixed alive in ethanol. found in the present study. In total, 6 different ascaridoid larval types were found, belonging to the genera Anisakis, Hysterothylacium, 2.2. Morphological examination of parasites Raphidascaris and Terranova (Table 2).Therewerealsoanumberoflar- vae that were damaged and could not be identified due to their A small piece of the mid-body of each larva was excised for molecu- condition. The description of these larval types and their genetic charac- lar study and the rest of the nematode was cleared in lactophenol for terisation are provided below. morphological examination. All parasite larvae were classified under In the host section below: distinct groups based on the morphology of the lips, digestive and ex- cretory systems and tail [15, 16]. A number of representatives from a indicates those fish for which their parasite specimens were identi- each group were then selected for a detailed measurement of the bodily fied morphologically and measured and their accession number features. All measurements are given in millimetres, unless stated oth- mentioned under “materials examined”; erwise. Mean measurements are given, followed by the range and num- b indicates those fish for which parasite specimens were assigned to ber of measured specimens in parentheses. Drawings were made using that morphotype based on the ITS-2 sequence; a microscope equipped with a drawing tube. All larval types in the pres- c indicates those fish for which parasite specimens were morphologi- ent study were washed in saline, then fixed in 70% ethanol and deposit- cally classified as the same morphotype (without detailed measure-