Molecular and Cellular Probes 21 (2007) 386–390 The specific identification of anisakid larvae from fishes from the Yellow Sea, China, using mutation scanning-coupled sequence analysis of nuclear ribosomal DNA $ Luping Zhang a,b , Min Hu a , Shokoofeh Shamsi a , Ian Beveridge a , Huimin Li b , Zhen Xu b , Liang Li b , Cinzia Cantacessi a , Robin B. Gasser a,Ã a Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei Province 050016, People’s Republic of China Received 17 March 2007; accepted 9 May 2007 Available online 21 May 2007 Abstract Nematodes of the family Anisakidae parasitize fish, mammals, birds and reptiles, with the larval stages of some species causing severe clinical disease in humans. Therefore, the accurate identification of anisakid nematodes is a key component of disease surveillance and control. An epidemiological survey of 123 fishes comprising eight different species from the Yellow Sea in China revealed that more than 25% of fish were infected with the larvae of anisakids, 200 third-stage larvae (L3s) were collected from fish and then subjected to morphological and molecular study. Larvae identified as Anisakis type I (n ¼ 197) and Hysterothylacium sp. (n ¼ 3), based on morphological criteria were characterized genetically by mutation scanning, followed by targeted sequencing of the first and second internal transcribed spacers of nuclear ribosomal DNA. Comparison of the sequences obtained from a subset of 27 specimens with those available in public gene databases showed that all samples identified morphologically as Anisakis type I (n ¼ 197) were Anisakis pegreffii, whereas those identified as Hysterothylacium sp. (n ¼ 3) were Hysterothylacium aduncum. The approach used herein was an effective means of matching incompletely identifiable larval nematodes with identifiable reference sequences, and provides a basis for exploring the composition of populations of anisakid larvae in fish as well as their ecology, particularly their life cycles and transmission patterns. Crown Copyright r 2007 Published by Elsevier Ltd. All rights reserved. Keywords: Nematodes; Anisakidae; Specific identification; Ribosomal DNA; Polymerase chain reaction (PCR); Single-strand conformation polymorphism (SSCP) Nematodes of the family Anisakidae parasitize fish, marine mammals, birds and reptiles [1]. While the life cycles of many species within the family remain unclear, it is known that species of marine fish can act as intermediate, paratenic or definitive hosts. It is also well recognized that some anisakids, such as Anisakis simplex, are transmissible to humans, where they can cause significant clinical disease [2–4]. Since the first reports confirming the pathogenic effects of Anisakis species in humans [5,6], there has been increasing awareness of fish-borne parasitic diseases [2,3,7,8]. The accurate identification of anisakid nematodes at any life cycle stage and in any host is central to understanding their ecology and epidemiology, diagnosis and is a key component of disease surveillance and control. In many clinical cases, there are considerable limitations associated with the specific identification of larval nematodes, due to difficulties in identifying larval stages using morphological characters [2,3,8,9] or when only small sections of worms are available for identification [3]. Polymerase chain reaction (PCR)-coupled methods can overcome such problems in that they allow the specific detection of parasite DNA from minute (nanogram to picogram) quantities of material [10], provided an appropriate DNA ARTICLE IN PRESS www.elsevier.com/locate/ymcpr 0890-8508/$ - see front matter Crown Copyright r 2007 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.05.004 $ Nucleotide sequence data reported in this paper are available in the GenBank database (via http://www.ncbi.nlm.nih.gov/) under accession nos. AM503953-AM503956. Ã Corresponding author. Tel.: +613 97312000; fax: +613 97312366. E-mail address: robinbg@unimelb.edu.au (R.B. Gasser).