Mutation scanning-coupled sequencing of nuclear ribosomal DNA spacers as a tool for the specific identification of different Contracaecum (Nematoda: Anisakidae) larval types

Abstract

In the present study, various morphotypes of Contracaecum (Nematoda) larvae in different developmental stages were described and then genetically characterised using sequence data obtained from the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The alignment of ITS-1 and ITS-2 sequence data for larvae investigated with those from well characterised adults in previous studies showed that fourth-stage Contracaecum larvae from the Australian pelican, Pelecanus conspicillatus, had the same sequence as Contracaecum microcephalum reported from the little pied cormorant, Phalacrocorax melanoleucos. In addition, four different morphotypes of third-stage larvae, including types I to IV, were found in various species of fish. Contracaecum type I larvae were composed of two distict genotypes; those collected from mullet, Mugil cephalus, had the same sequence as C. multipapillatum, and those from an unknown species of hardyhead (family Atherinidae) were C. pyripapillatum. Contracaecum type II larvae from mackerel, Scomber australasicus, had the same sequence as adult C. ogmorhini sensu stricto, found in the Australian and New Zealand fur seals ( Arctocephalus spp). Contracaecum type III larvae from flathead, Platycephalus laevigatus, had the same sequence as C. rudolphii D found in the black cormorant, Phalacrocorax carbo. The approach used herein was an effective means of matching incompletely identifiable larval nematodes with 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.