A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of the Anisakis simplex (s. l.) complex (Nematoda, Anisakidae).

Marialetizia Palomba,Simonetta Mattiucci,Michela Paoletti,Giuseppe Nascetti,Stephen C. Webb

doi:10.1051/parasite/2020033

Marialetizia Palomba, Simonetta Mattiucci + Show 3 more

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https://doi.org/10.1051/parasite/2020033

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Abstract

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

Highlights

The Anisakis simplex (s. l.) species complex includes heteroxenous nematodes belonging to three distinct species, namely A. pegreffii, A. simplex (s. s.) and A. berlandi [25]
While it was widely demonstrated that A. simplex (s. s.) and A. pegreffii [6, 13, 14] infect the musculature of the fish hosts, there is no knowledge about the ability of A. berlandi to invade the fish host musculature
According to the sequences of 629 bp in length of the mtDNA cox2 gene locus [22], 76 specimens were assigned to A. pegreffii, 93 to the species A. simplex (s. s.) and, 50 individuals corresponded to A. berlandi

Summary

Introduction

The Anisakis simplex (s. l.) species complex includes heteroxenous nematodes belonging to three distinct species, namely A. pegreffii, A. simplex (s. s.) and A. berlandi [25]. L.) species complex includes heteroxenous nematodes belonging to three distinct species, namely A. pegreffii, A. simplex The geographical range of these three species is quite distinct; some oceanographic basin waters are reported as overlapping areas and contact zones between them (for a review, [25]). In allopatric and sympatric areas, they are known to infect several definitive and intermediate/paratenic hosts; the three species often being found parasitizing the same host-species range [25]. Humans are accidental hosts who become infected after the ingestion of raw fish and squids containing, in their edible parts, the third-stage larvae (L3) of A. pegreffii and A. simplex S.) and A. pegreffii [6, 13, 14] infect the musculature of the fish hosts, there is no knowledge about the ability of A. berlandi to invade the fish host musculature While it was widely demonstrated that A. simplex (s. s.) and A. pegreffii [6, 13, 14] infect the musculature of the fish hosts, there is no knowledge about the ability of A. berlandi to invade the fish host musculature

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