Characterisation of Ascaridoid Larvae from Marine Fish Off New Caledonia

Parasitology International 64 (2015) 397–404

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Parasitology International

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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

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 specimens 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 excretory 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- ed in the Museum National d'Histoire Naturelle, Paris France. Museum ments due to the condition of these specimens); registration numbers are also provided. d indicates those fish for which parasite specimens presented the same size for the ITS-2 amplicon. 2.3. Molecular study All specimens from the same morphotype presented the same band 2.3.1. DNA extraction and PCR size on agarose gel and each was distinct from the others both morpho- Genomic DNA (gDNA) was isolated from all individual larvae by so- logically and based on the band size. Further details are provided under dium dodecyl-sulphate/proteinase K treatment, column-purified each taxa. (Wizard™ DNA Clean-Up, Promega) and eluted into 45 μlofwater. PCR was used to amplify the ITS-2 region using primers (SS2: 5′-TTGC 3.1. Anisakis larval type I of Cannon, 1977 (Fig. 1a&b) AGACACATTGAGCACT-3′ (forward) and NC2: 5′-TTAGTTTCTTTTCCTC CGCT-3′ (reverse)) and cycling conditions, initial 94 °C/5′,then94°C/ 3.1.1. Materials examined 30″,55°C/40″, 72 °C/40″ × 30 cycles, 72 °C/5 extension and 4 °C. An al- JNC2995-1, JNC2995-2, JNC2996-2, JNC2996-3, JNC3000-1, iquot (4 μl) of each amplicon was examined on a 1.5% w/v agarose gel, JNC3003-4, JNC3004-5, JNC3006-10, JNC3006-5, JNC3007-1, JNC3009- S. Shamsi et al. / Parasitology International 64 (2015) 397–404 399

Table 2 Details about ﬁsh, including MNHN JNC number, species, family, date of collection, locality, fork length and weight. Alphabetical order of families and species.

Family Species MNHN Date Locality Fork length Weight Ascaridoid larvae found in the present JNC (mm) (g) study

Balistidae Abalistes stellatus 2914 21/04/2009 Off Récif Toombo, 20–40 m depth 470 2000 Hysterothylacium type XIII Carangidae Alepes vari 3191 25/06/2010 Nouméa Fishmarket 283 350 Unidentified larval ascaridoid Carangidae Atule mate 2964 05/06/2009 Nouméa Fishmarket 412 334 Unidentified larval ascaridoid Carangidae Atule mate 2965 05/06/2009 Nouméa Fishmarket 315 519 Unidentified larval ascaridoid Carangidae Atule mate 2909 17/04/2009 Nouméa Fishmarket 231 201 Terranova type II ⁎ Carangidae Carangoides chrysophrys 3179 03/06/2010 Nouméa Fishmarket 245 290 Anisakis larval type I ⁎ Carangidae Carangoides chrysophrys 3212 21/07/2010 Nouméa Fishmarket 265 398 Anisakis larval type I Raphidascaris larval type ⁎ Carangidae Carangoides chrysophrys 3210 21/07/2010 Nouméa Fishmarket 285 446 Raphidascaris larval type ⁎ Carangidae Carangoides chrysophrys 3211 21/07/2010 Nouméa Fishmarket 265 360 Raphidascaris larval type Carangidae Carangoides fulvoguttatus 3180 03/06/2010 Nouméa Fishmarket 295 430 Terranova type II Carangidae Carangoides fulvoguttatus 3218 05/08/2010 Nouméa Fishmarket 273 348 Terranova type II Carangidae Carangoides hedlandensis 3172 27/05/2010 Nouméa Fishmarket 245 341 Anisakis larval type I Carangidae Carangoides orthogrammus 3195 08/07/2010 Nouméa Fishmarket 340 842 Terranova type II ⁎ Carangidae Carangoides cf talamparoides 3243 14/09/2010 Nouméa Fishmarket 270 425 Anisakis larval type I Terranova type II Carangidae Selar crumenophtalmus 3044 10/09/2009 Nouméa Fishmarket 228 209 Unidentified larval ascaridoid Carangidae Selar crumenophtalmus 3043 10/09/2009 Nouméa Fishmarket 227 194 Anisakis larval type I Chirocentridae Chirocentrus dorab 3236 09/09/2010 Nouméa Fishmarket 700 1538 Anisakis larval type I Chirocentridae Chirocentrus dorab 3222 13/08/2010 Nouméa Fishmarket 490 515 Raphidascaris larval type Chirocentridae Chirocentrus dorab 3219 11/08/2010 Nouméa Fishmarket 535 723 Terranova type II Clupeidae Herklotsichthys quadrimaculatus 2948 27/05/2009 Nouméa Fishmarket 126 26.6 Hysterothylacium type XIV Diodontidae Diodon hystrix 3199 08/07/2010 Nouméa Fishmarket 380 1900 Raphidascaris larval type Elopidae Elops cf hawaiensis 3252 16/09/2010 Nouméa Fishmarket 450 815 Anisakis larval type I Lethrinidae Gymnocranius euanus 3065 30/09/2009 Off Récif Toombo, 20–40 m depth 294 528 Hysterothylacium larval type VI Lethrinidae Gymnocranius superciliosus 3064 30/09/2009 Off Récif Toombo, 20–40 m depth 330 708 Hysterothylacium larval type VI Lutjanidae Pristipomoides argyrogrammicus 2995 07/07/2009 Off Récif Toombo, 250 m depth 255 323 Anisakis larval type I Lutjanidae Pristipomoides argyrogrammicus 2996 07/07/2009 Off Récif Toombo, 250 m depth 190 127 Anisakis larval type I Malacanthidae Branchiostegus wardi 2997 07/07/2009 Off Récif Toombo, 250 m depth 383 645 Raphidascaris larval type Monodactylidae Monodactylus argenteus 3215 23/07/2010 Nouméa Fishmarket 170 123 Anisakis larval type I Terranova type II Nemipteridae Pentapodus nagasakiensis 2976 11/06/2009 Off Ile Redika, ca. 20 m depth 156 65 Anisakis larval type I Scombridae Rastrelliger kanagurta 3000 06/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3003 06/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3004 06/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3006 07/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3007 07/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3009 07/08/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Scombridae Rastrelliger kanagurta 3002 06/08/2009 Nouméa Fishmarket N/A N/A Hysterothylacium larval type VI Terranova type II Scombridae Rastrelliger kanagurta 3005 06/08/2009 Nouméa Fishmarket N/A N/A Hysterothylacium type XIV Scombridae Scomberomorus commerson 3177 28/05/2010 Nouméa Fishmarket N/A N/A Terranova type II Serranidae Epinephelus coioides 3257 23/09/2010 Nouméa Fishmarket N/A N/A Serranidae Epinephelus coioides 3140 Anisakis larval type I Serranidae Epinephelus maculatus 3031 01/09/2009 Off Récif Toombo 387 716 Terranova type II Sphyraenidae Sphyraena forsteri 3198 08/07/2010 Nouméa Fishmarket 490 727 Anisakis larval type I Hysterothylacium larval type VI Terranova type II Sphyraenidae Sphyraena putnamae 3035 01/09/2009 Off Récif Toombo 660 1650 Anisakis larval type I Terranova type II Synodontidae Saurida undosquamis 3132 Anisakis larval type I

Trichiuridae Trichiurus lepturus 3046 10/09/2009 Nouméa Fishmarket N/A N/A Unidentiﬁed larval ascaridoid Trichiuridae Trichiurus lepturus 3047 10/09/2009 Nouméa Fishmarket N/A N/A Unidentiﬁed larval ascaridoid Trichiuridae Trichiurus lepturus 3045 10/09/2009 Nouméa Fishmarket N/A N/A Anisakis larval type I Hysterothylacium type XIV Terranova type II Trichiuridae Trichiurus lepturus 3159 30/04/2010 Nouméa Fishmarket 700 N/A Anisakis larval type I

⁎ See Materials and methods section for remarks on identiﬁcation.

1, JNC3045-2, JNC3045-3, JNC3045-5, JNC3159-2, JNC3159-5, JNC3172- 0.011; n = 20) of body length. Rectum short, oblique to anus, with a 5, JNC3198-1, JNC3236-4, JNC3236-5. thick cuticular layer. Tail short, conical, with rounded tip, ending in a single mucron. 3.1.2. Description Third stage larvae. Body length 18.51 (9.65–24.85; n = 20), width 0.45 (0.36–0.60; n = 20). Labia poorly developed. Tooth present. Excre- 3.1.3. Hosts tory pore immediately below tooth. Nerve ring 0.15 (0.06–0.35; n = 15) Carangoides chrysophrysc,d, C. fulvoguttatusc,d, C. hedlandensisa,c,d, from anterior end. Oesophagus muscular, ends in ventriculus, 1.69 Carangoides cf talamparoidesc,d, Chirocentrus doraba,c,d, Elops cf. (1.23–2.23; n = 20) long, 0.01 (0.06–0.22; n = 20) of body length. Ven- hawaiensisb,c,d, Epinephelus coioidesb,c,d, Monodactylus argenteusc,d, triculus obliquely joined with intestine, 0.92 (0.69–1.28; n = 20) long. Pentapodus nagasakiensisc,d, Pristipomoides argyrogrammicusa,b,c,d, Anus 0.10 (0.05–0.15; n = 20) from posterior end, 0.006 (0.002– Rastrelliger kanagurtaa,c,d, Saurida undosquamisc,d, Selar 400 S. Shamsi et al. / Parasitology International 64 (2015) 397–404

Fig. 1. Ascaridoid larvae found in the present study: a & b, anterior and posterior ends of Anisakis larval type 1, respectively, c & d, anterior and posterior ends of Hysterothylacium type VI, respectively, e & f, anterior and posterior ends of Hysterothylacium type XIII, respectively, g & h, anterior and posterior ends of Hysterothylacium type XIV, respectively, i & j, anterior and posterior ends of Rhidascaris type larva, respectively, k & l, anterior and posterior ends of Terranova type II, respectively (scale bars 100 μ). crumenophthalmusc,d, Sphyraena forsteria,c,d, S. putnamaeb,c,d, Trichiurus demersal species that can inhabit a range up to 1075 m deep in the lepturusa,b,c,d. ocean.

3.1.4. Location 3.2. Hysterothylacium larval type VI of Shamsi, Gasser, Beveridge 2013 Intestinal lumen and abdominal organs. (Fig.1c&d)

3.1.5. Genetic characterisation 3.2.1. Materials examined Six representatives were selected for amplification of the ITS-2 re- JNC3064-1 to 6, JNC3065-1 to 3, JNC3198-3, JNC3002-10. gion of rDNA. The length of the ITS-2 was 355 bp. It was identical amongst all specimens belong to this morphotype. No sequence poly- 3.2.2. Description morphism was detected. Sequences of the ITS-2 region of specimens Third stage larvae, very small, labia not developed (Fig. 1c), excreto- – in the present study (accession numbers LN651095 LN651100) were ry pore between tip of intestinal caecum and nerve ring (closer to the identical to the ITS-2 sequence of A. typica (GenBank accession number: intestinal caecum), ventriculus round, intestinal caecum shorter than AY826724). ventricular appendix, ventricular appendix almost same size as oesophagus, intestine with sinusoidal pattern (Fig. 2a), tail relatively long, con- 3.1.6. Ecological remarks ical, slightly swollen at tip (Fig. 2b), tapering ratio 2–2.3:1, tail's tip The list of hosts includes several fish which are characteristic of the rounded with a single minute spine, visible in high magnification lagoon of New Caledonia, including pelagic–neritic fish such as (Fig. 2b). Rastrelliger kanagurta, reef-associated fish such as S. crumenophthalmus Body length 4.78 (3.13–6.50; n = 9). Body width 0.24 (0.15–0.33; and Chirocentrus dorab, benthic reef-associated fish such as Saurida n = 8). Nerve ring 0.22 (0.18–0.28; n = 8) from anterior end. Excretory undosquamis and Synodus sp. and, curiously, a deep-sea fish from off pore 0.32 (0.20 –0.44; n = 7) from anterior end. Oesophagus 0.48 (0.38– the barrier reef, Pristipomoides argyrogrammicus. It seems that Anisakis 0.61; n = 7) long, 0.09 (0.08–0.11; n = 7) of body length. Ventriculus typica shows a broad habitat range irrespective of the ecology of its 0.06 (0.05–0.08; n = 8) long. Ventricular appendix 0.48 (0.40–0.73; fish host. This is similar to the review by Mattiucci and Nascetti [17] n = 8) long, 2.08 (1.52–2.76; n = 7) times oesophageal length. Intesti- who reported the distribution range of 30°S to 35°N in warmer temper- nal caecum 0.14 (0.08–0.20; n = 8) long, 0.30 (0.21–0.43; n = 7) of oe- ate and tropical waters for A. typica, from a broad range of fish hosts sophageal length and 0.29 (0.18–0.50; n = 8) of ventricular appendix such as S. crumenophthalmus, which is a reef-associated species living length. Tail 0.14 (0.10–0.18; n = 8) long, 0.03 (0.02–0.04; n = 7) between 0 and 170 m water depth or Merluccius merluccius, which is a body length. S. Shamsi et al. / Parasitology International 64 (2015) 397–404 401

Fig. 2. a) Sinusoidal pattern of the intestine in Hysterothylacium larval type VI, b) shows tapering tail with a single spin at the tip, c) serpengenous pattern of the intestine in Hysterothylacium larval type XIII, d) tapering tail in Hysterothylacium larval type XIII, e) tapering tail in Hysterothylacium larval type XIV.

3.2.3. Hosts 3.2.6. Ecological remarks Gymnocranius euanusa,b,c,d, Gymnocranius superciliosusa,b,c,d, All four host species reported in the present study are coral-reef as- Rastrelliger kanagurtaa,c,d, Sphyraena forsteria,b,c,d. sociated ﬁsh from the lagoon. In a previous study [15], this larval type was also reported from a range of reef-associated ﬁsh belonging to the genus Chaetodon. 3.2.4. Location Intestinal lumen and abdominal organs. 3.3. Hysterothylacium type XIII (Fig. 1e&f)

3.3.1. Material examined 3.2.5. Genetic characterisation JNC2914-2. Three representatives were selected for amplification of the ITS-2 region of rDNA. The ITS-2 region was 272 bp long and identical amongst all specimens. No sequence polymorphism was detected. 3.3.2. Description Hysterothylacium larval type VI in the present study (accession num- Third stage larva. Labia not developed. Body length 9.88, width 0.31. bers LN651101–LN651103) had identical ITS-2 sequences with those Nerve ring 0.30 from anterior end. Excretory pore slightly anterior to previously described and characterised as Hysterothylacium larval anterior end of intestinal caecum, 0.62 from anterior end. Oesophagus type VI (accession numbers FN811701–FN811702) [15].Noidentical muscular, ends in ventriculus, 0.75 long, 0.076 of body length. Ventricu- sequence with known adults was found in GenBank. The closest lus subglobular, 0.05–0.15. Ventricular appendix 0.70 long, 0.93 of oe- similarity was with the ITS-2 sequence of Hysterothylacium sophagus length. Intestinal caecum 0.24 long, 0.32 of oesophagus deardorffoverstreetorum (accession number JF730204; 9 bp differ- length, 0.34 ventricular appendix length. Intestine with serpiginous pat- ence; 97% similarity). tern (Fig. 2c). Anus 0.02 from posterior end, 0.020 of body length. Tail 402 S. Shamsi et al. / Parasitology International 64 (2015) 397–404 rounded, minute fine single spine at tip (visible at higher magnification should be noted C. muraenesoxi is in fact Hysterothylacium muraenesoxi only), tapering ratio 1.2:1 (Fig. 2d). as proposed by Deardorff and Overstreet [19].

3.3.3. Host 3.4.6. Ecological remark a,b,c,d Abalistes stellatus . All three fish in the present study were caught from inside the lagoon, the first likely being prey for the two other larger species. Howev- 3.3.4. Location er, Trichiurus lepturus has a wider range of depth, being benthopelagic, Intestinal lumen and abdominal organs. with a 0–589 m depth range (http://www.fishbase.org/summary/ Trichiurus-lepturus.html accessed 7/11/2014). 3.3.5. Genetic characterisation fi The sole specimen in the present study was subjected to ampli cation 3.5. Raphidascaris larval type (Fig. 1i&j) of the ITS-2 region of rDNA. The length of the ITS-2 was 262 bp. No identical or highly similar ITS-2 sequence was found in the GenBank for this 3.5.1. Materials examined specimen (accession number LN651104). The highest similarity with a JNC2997-1 to JNC2997-2, JNC3211-1, JNC3212-1 to JNC3212-2, known adult was with ITS-2 sequence of H. deardorffoverstreetorum (ac- JNC3210-1 to JNC3210-2, JNC3222-1 to JNC3222-5. cession number JF730204; 20 bp difference; 90% similarity).

3.5.2. Description 3.3.6. Ecological remark Third stage larvae. Body length 5.49 (4.13–6.50; n = 10), width 0.28 A. stellatus is a benthic reef-associated ﬁsh, common in the lagoon of (0.25–0.38; n = 10). Labia slightly developed. Nerve ring 0.22 (0.18– New Caledonia. 0.28; n = 9) from anterior end. Excretory pore below nerve ring 0.33 (0.27–0.43; n = 5) from anterior end. Oesophagus muscular, ends in 3.4. Hysterothylacium type XIV (Fig.1g&h) ventriculus, 0.67 (0.48–0.90; n = 10) long, 0.12 (0.10–0.14; n = 8) of body length. Ventriculus oblong, 0.07 × 0.09 (0.04–0.11 × 0.05–0.15; 3.4.1. Materials examined n = 7). Anus 0.21 (0.10–0.35; n = 7) from posterior end, 0.04 (0.02– JNC3005-10, JNC2948-2 to JNC2948-7. 0.05; n = 7) of body length.

3.4.2. Description Third stage larvae. Cuticle with delicate annulation. Labia not devel- 3.5.3. Hosts a,b,c,d a,b,c,d oped (Fig. 1g). Body length 4.93 (3.88–5.75; n = 6), maximum body Branchiostegus wardi , Carangoides chrysophrys , Chirocentrus a,c,d c,d width 0.18 (0.15–0.25; n = 6). Nerve ring 0.27 (0.22–0.33; n = 5) dorab , Diodon hystrix from anterior end. Excretory pore between nerve ring and intestinal caecum's tip (closer to nerve ring), 0.37 (0.31–0.43; n = 2) from ante- 3.5.4. Location rior end. Oesophagus muscular, ends in ventriculus, 0.66 (0.33–0.86; Intestinal lumen and abdominal organs. n = 5) long, 0.13 (0.06–0.16; n = 5) of body length. Ventriculus 0.10 – – (0.10 0.10; n = 2) long. Ventricular appendix 0.39 (0.24 0.54; n = 5) 3.5.5. Genetic characterisation – long, 0.60 (0.39 0.81; n = 5) of oesophagus length. Intestinal caecum Two representatives were selected for ampliﬁcation of the ITS-2 re- – – 0.18 (0.13 0.26; n = 5) long, 0.28 (0.21 0.38; n = 5) oesophagus gion of rDNA. ITS-2 had 287 bp and was identical amongst specimens. – length and 0.48 (0.35 0.63; n = 5) ventricular appendix length. Anus No sequence polymorphism was detected in the ITS-2. Sequences of – – 0.18 (0.10 0.28; n = 7) from posterior end, 0.037 (0.026 0.055; n = the ITS-2 region of specimens in the present study (accession numbers – 5) of body length. Tail tip bent dorsally ending to 3 4 sharp triangular LN651108–LN651109) were identical with the ITS-2 sequence of minute conical spines (Fig. 2e). Raphidascaris trichiuri (GenBank accession number: FJ009682) [20].

3.4.3. Hosts 3.5.6. Ecological remark Herklosichthys quadrimaculatusa,b,c,d, Rastrelliger kanagurtaa,b,c,d and The list of fish includes a curious assemblage of species from inside Trichiurus lepturusb,c,d. the lagoon and Branchiostegus wardi, a demersal fish from the deep- sea off the barrier reef. 3.4.4. Location Intestinal lumen and abdominal organs. 3.6. Terranova type II of Cannon, 1977 (Fig. 1k&l) 3.4.5. Genetic characterisation Three representatives were selected for amplification of the ITS-2 re- 3.6.1. Materials examined gion of rDNA. ITS-2 in these specimens was 276 bp long. One specimen JNC3195, JNC2909-10, JNC2909-2, JNC2909-6, JNC2909-8, JNC3002- had one base pair difference (transitional mutation) with the other 5, JNC3002-6, JNC3002-9, JNC3045-10, JNC3177-2, JNC3177-4, specimens in alignment position 236. Other than this base pair differ- JNC3177-5, JNC3177-7, JNC3215-7, JNC3215-8, JNC3243-1. ence in one specimen, sequences of the ITS-2 region of specimens in the present study (accession numbers LN651105–LN651107) when 3.6.2. Description compared with those deposited in the GenBank database showed that Third stage larvae. Cuticle with delicate annulation. Body length 4.87 they are identical to accession number JX848692 reported previously (2.45–9.25; n = 16), width 0.24 (0.16–0.35; n = 16). Labia poorly de- from Lizard Island, Australia [18]. We suggest that JX848692 was a mis- veloped. Tooth present. Excretory pore immediately below tooth. identification in that study and should be classified as Hysterothylacium Nerve ring 0.07 (0.05–0.09; n = 7) from anterior end. Oesophagus mus- type XIV and not as Hysterothylacium type X (for further details please cular, ends in large oval ventriculus, 0.61 (0.45–0.77; n = 16) long, 0.14 see Discussion section). The highest similarity with a known adult was (0.05–0.23; n = 16) of body length. Ventriculus 0.28 (0.22–0.45; n = with the ITS-2 sequence of Contracaecum muraenesoxi (accession num- 16) long. Anus 0.12 (0.09–0.16; n = 14) from posterior end, 0.03 ber EU828749; 32 bp difference; 87% similarity) and H. zhoushanensis (0.02–0.04; n = 14) of body length. Tail strongly annulated, conical, ta- (accession number JX848692; 33 bp difference; 86% similarity). It pering smoothly. S. Shamsi et al. / Parasitology International 64 (2015) 397–404 403

3.6.3. Hosts publication [23] in which a sequence had been deposited in GenBank Alepes varib,c,d, Atule matea,c,d, Carangoides fulvoguttatusc,d, with an identical sequence to Hysterothylacium type XIV in the Carangoides orthogrammusa,c,d, Carangoides cf. talamparoidesa,c,d, present study, however it has been inaccurately assigned it to the Chirocentrus dorabb,c,d, Epinephelus maculatusc,d, Monodactylus original Hysterothylacium type X of Shamsi, 2007 [15]. argenteusa,b,c,d, Rastrelliger kanagurtaa,b,c,d, Sphyraena forsteric,d, Raphidascaris larval type in the present study was identified as Sphyraena putnamaec,d, Scomberomorus commersona,c,d, Trichiurus R. trichiuri. This is the first report of occurrence of this species in New lepturusa,c,d. Caledonian waters. Apart from Raphidascaris and Anisakis larval types which were identified to the species level based on matching their 3.6.4. Location ITS-2 sequences with well identified adults, the specificidentityof Intestinal lumen and abdominal organs. other ascaridoid larvae found in the present study remain unknown. The lack of a match between sequences of these ascaridoid larvae with 3.6.5. Genetic characterisation known species is very interesting, suggesting that the adult stage of 11 representatives were selected for amplification of the ITS-2 re- these parasites await discovery and shows the huge potential for future gion of rDNA. ITS-2 was 252 bp. Sequence polymorphism was detected research on these economically important parasites. in alignment position 22. Sequences of the ITS-2 region of specimens in This is the first report, describing and genetically characterising the present study (accession numbers LN651110–LN651120) were ascaridoid larval types from New Caledonian waters. Although some identical to ITS-2 sequence of Terranova Type II of Shamsi (accession studies showed that both first and second internal transcribed spacers numbers: LN795872 and LN795853). No similarity was observed be- (ITS-1 and ITS-2, respectively) can be useful for specific identification tween ITS-2 sequences of the specimens in the present study and any of ascaridoid larvae e.g., [15, 24] only ITS-2 was used in the present known adults belonging to the family Anisakidae. study due to the significantly lower level of nucleotide variation reported for ITS-1 compared to ITS-2 [15] as well as no variation reported in 3.6.6. Ecological remarks the ITS-1 of some of the distinct but closely related taxa. For example, All fish listed above are from inside the lagoon, but T. lepturus has a two members of the C. osculatum sensu lato, C. osculatum Dand wider range of depth. These Terranova larval types have been reported C. osculatum E, for which multilocus enzyme electrophoresis data from a broad range of fish, including Apogonidae, Atherinidae, showed these taxa are genetically distinct from one another and from Cirrhitidae, Lethrinidae, Lutjanidae, Chaetodontidae, Pseudochromidae, other members of the C. osculatum sensu lato [25] showed no nucleotide Scombridae, Serranidae, Nemipteridae, Carangidae and Sphyraenidae difference between their ITS-1 sequences [26, 27]. Nevertheless, as from Australian waters. There are also some early reports of Terranova shown in several studies [e.g., 24], combined ITS-1 and ITS-2 sequences Type II (based on morphology only) in Queensland waters, by Cannon can be useful for specific identification of larvae and future studies on [21] who found Terranova larval types only in predators of nektons the ITS-1 of larval found in the present study might further elucidate and in an intermediate water depth. their taxonomic status. Given that out of hundreds species of fish inhabiting New Caledo- 4. Discussion nian waters only 28 species were examined in the present study and six different larval types were found, it is expected that ascaridoid larvae One important finding of the present study is the specificidentifica- be highly diverse in New Caledonian waters. It is also noteworthy that tion of Anisakis larval type I as A. typica. Anisakis larvae are known to be we selected representatives from each morphotype to be genetically one of the main causes of anisakidosis worldwide; however, so far most characterised based on their ITS-2 sequence. Therefore, there might be human cases seem to be due to infection with members of A. simplex more genotypes than what has been reported in the present study. sensu lato [22]. Although fish infected with A. typica can potentially be regarded as a source of human anisakidosis, so far infection with Acknowledgement A. typica has not been confirmed in humans. To our knowledge, no case of human anisakidosis has been reported in New Caledonia al- This work was funded by Charles Sturt University (Honours fund by fi though the consumption of raw sh is widespread in the population. School of Animal and Veterinary Sciences and Faculty of Science Seed fi Apart from S. crumenophthalmus and Sphyraena forsteri, all other sh Grant). The following students helped for parasite collection: Adeline hosts reported for A. typica in the present study are being recorded for Grugeaud, Marine Briand, and Charlotte Schoelinck. Fish identification fi the rst time. was helped by the following ichthyologists: Brian Russel (Pentapodus), fi Another important nding of the present study is describing two Samuel Iglésias (MNHN), Bernard Séret (IRD/MNHN). We thank the new Hysterothylacium larval types XIII and XIV. Previously, 12 differ- anonymous reviewers for their helpful suggestions. We also thank Mr ent morphotypes of Hysterothylacium were described [15,16]. Richard Carroll for editing the manuscript. Hysterothylacium type XIII is most similar to Hysterothylacium type VI in the morphology of the tail ornament and ratio of the intestinal References caecum to the ventricular appendix. Hysterothylacium type XIII can be morphologically differentiated from Hysterothylacium type VI by [1] J.W. Smith, Ascaridoid nematodes and pathology of the alimentary tract and its as- having a globular ventriculus. In addition, these two genetically dis- sociated organs in vertebrates, including man: a literature review, Helminthol Abstr 68 (1999) 49–96. tinct larval types could be morphologically differentiated based on [2] P.H. Van Thiel, Anisakiasis, Parasitology 52 (1962) 16–17. the difference in body length (9.88 mm and 3.13–6.50 mm for XIII [3] S. Shamsi, Recent advances in our knowledge of Australian anisakid nematodes, Int J and VI, respectively) and tapering ratio of the tail (1.2: 1 in type Parasitol Parasites Wildl 3 (2014) 178–187. fi XIII versus 2–2.3: 1 in type VI). The other new morphotype in the [4] J.L. Justine, Parasites of coral reef sh: how much do we know? With a bibliography of fish parasites in New Caledonia, Belg J Zool 140 (Suppl.) (2010) 155–190. present study, Hysterothylacium type XIV is most similar to [5] F. Moravec, J.L. 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