Genetic characterisation and taxonomy of species of Anisakis (Nematoda : Anisakidae) parasitic in Australian marine mammals Shokoofeh Shamsi A,C , Robin Gasser B and Ian Beveridge B A School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Estella, NSW 2678, Australia. B Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Vic. 3030, Australia. C Corresponding author. Email: sshamsi@csu.edu.au Abstract. Three species of Anisakis from Australian marine mammals, including Anisakis brevispiculata, A. simplex C and A. pegreffii, are described and characterised genetically on the basis of sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear rDNA. Parasite specimens were collected from Delphinus delphis, Tursiops truntatus and Kogia sima in Australia. A. brevispiculata is reported for the first time in Australia. However, analyses of sequence data suggests that A. brevispiculata in Australia is genetically distinct from specimens considered to represent the same species from other parts of the world. Fourth larval and adult stages of A. pegreffii were found in dolphins. Assigning larvae to A. pegreffii was based on the ITS-1 and ITS-2 sequences. A description of these larvae also is provided. Furthermore, fourth-stage larvae of A. simplex C were found in Kogia sima. Alignments of ITS-1 and ITS-2 sequences for members of A. simplex sensu lato revealed that nucleotide differences in ITS-1 can be used to differentiate among members of A. simplex sensu lato. This study reinforces the use of a combined molecular and morphological approach for the specific identification of anisakid nematodes. Received 11 May 2011, accepted 6 February 2012, published online 6 August 2012 Introduction Anisakid nematodes have complex life-cycles that involve various invertebrates (crustaceans and/or molluscs [squids]) and fishes as intermediate/paratenic hosts and marine mammals as definitive hosts (Anderson 2000). Species of Anisakis are the most abundant within the family and some of them are infective to humans (Cheng 1982). Although these parasites can result in heavy infections in marine mammals, there is disagreement concerning their pathogenicity. Whilst some authors believe that infections with anisakid nematodes are insignificant in marine mammals (Geraci and St Aubin 1987), other workers state that anisakids can be quite harmful in marine mammals (Stroud and Roffe 1979; McColl and Obendorf 1982; Jefferies et al. 1990; Abollo et al. 1998). One of the reasons for this disagreement might be due to difficulties in specific identification based on morphological criteria as well as the occurrence of sibling species within a morphospecies, with different sibling species exhibiting differing levels of pathogenicity (Nascetti et al. 1983; Paggi et al. 2001; Mattiucci and Nascetti 2006). Specific identification and the number of species within Anisakis have changed over the last few decades. In his revision of this genus, Davey (1971) considered only A. simplex (Rud., 1809, det. Krabbe, 1878), A. typica (Diesing, 1860) and A. physeteris Baylis, 1923 as valid species. Subsequently, genetic analysis showed that A. simplex comprised three genetically distinct species, including A. pegreffii Campana- Rouget & Biocca, 1955, A. simplex sensu stricto (Rudolphi, 1809) and A. simplex C Mattiucci, Nascetti, Cianchi, Paggi, Arduino, Margolis, Brattey, Webb, D’Amelio, Orecchia & Bullini, 1997 (Nascetti et al. 1986; Mattiucci et al. 1997). Later, A. brevispiculata Dollfus, 1966, which was earlier synonymised with A. physeteris (Davey 1971), and A. schupakovi Mozgovoi, 1951, which was considered as inquienda by Davey (1971), proved to be valid species (D’Amelio et al. 2000; Mattiucci et al. 2001). Moreover, two new species (A. ziphidarum Paggi, Nascetti, Webb, Mattiucci, Cianchi & Bullini, 1998 and A. paggiae Mattiucci, Nascetti, Dailey, Webb, Barros, Cianchi & Bullini, 2005) were detected within the genus using molecular approaches (Paggi et al. 1998; Mattiucci et al. 2005). Unlike the situation in the Northern Hemisphere, Anisakis species have been poorly studied in the Southern Hemisphere. Reports of anisakid nematodes from Australian marine mammals are very limited. Approximately eight species of anisakid parasites were reported from Australian mammals (Mackerras 1958). However, on the basis of Davey’s(1971) revision, only two species can be considered as valid species, and the other species remain in doubt. Therefore, Anisakis is currently represented by two species in Australia: A. simplex sensu lato (Rudolphi, 1809) reported from pinnipeds (the leopard Journal compilation Ó CSIRO 2012 www.publish.csiro.au/journals/is CSIRO PUBLISHING Invertebrate Systematics, 2012, 26, 204–212 http://dx.doi.org/10.1071/IS11019