The specific identification of anisakid larvae from fishes from the Yellow Sea, China, using mutation scanning-coupled sequence analysis of nuclear ribosomal DNA

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=197) and Hysterothylacium sp. (n=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=197) were Anisakis pegreffii, whereas those identified as Hysterothylacium sp. (n=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.