Novel polymorphic microsatellite loci inAnisakis pegreffiiandA. simplex(s. s.) (Nematoda: Anisakidae): implications for species recognition and population genetic analysis

Abstract

The species of Anisakis constitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in the A. simplex (s. l.) complex, i.e. A. pegreffii, A. simplex (s. s.) and A. berlandi. They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784, Anisl 08059, Anisl 00875, Anisl 07132, Anisl 00314, Anisl 10535 and Anisl 00185), were developed and validated on a total of N = 943 specimens of A. pegreffii and A. simplex (s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locus Anisl 7, previously detected in those Anisakis spp., was investigated. The parasites were first identified by sequence analysis of the EF1 α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two species A. pegreffii, A. simplex (s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in both Anisakis species and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

Cross-species utility of microsatellite loci for the genetic characterisation ofAnisakis berlandi(Nematoda: Anisakidae)

“…However, among the nuclear markers, those inferred from allozymes, despite their utility in the discovery of anisakid species and their genetic identification at any lifehistory stage, are not a standardised approach and are not available in all laboratories for species recognition. Conversely, DNA microsatellites have recently become the alternative nuclear markers of choice to be used for species recognition and population genetic analysis of nematodes included in the A. simplex (s. l.) complex [8]. Some DNA microsatellite loci were developed in A. pegreffii and A. simplex (s. s.) [15]; however, no diagnostic loci between these taxa were evidenced in the study.…”

“…Nematodes collected from the stomach of their hosts were washed in saline solution and then preserved, frozen at À80°C, as part of the collection of anisakids stored at the Department of Public Health and Infectious Diseases of Sapienza -University of Rome. Because of our previous findings of microsatellites among those developed as sex-linked loci [8], only adult nematodes were used for the genetic characterisation of the species based on SSR loci. Thus, the nematodes were first distinguished as L4-stage larvae and adults; then female and male adults were selected, according to the main morphological features that are diagnostic between sexes [10].…”

“…Some DNA microsatellite loci were developed in A. pegreffii and A. simplex (s. s.) [15]; however, no diagnostic loci between these taxa were evidenced in the study. Conversely, more recently, novel DNA microsatellite loci discovered in the species A. pegreffii were found to crossamplify the species A. simplex (s. s.) [8]. They were also found to be of diagnostic value in the recognition of A. pegreffii and A. simplex (s. s.), and for their population genetics analysis [8].…”

“…Conversely, more recently, novel DNA microsatellite loci discovered in the species A. pegreffii were found to crossamplify the species A. simplex (s. s.) [8]. They were also found to be of diagnostic value in the recognition of A. pegreffii and A. simplex (s. s.), and for their population genetics analysis [8].…”

First Molecular Diagnosis of Clinical Cases of Gastric Anisakiosis in Spain

“…In the last two decades, the use of molecular methods as PCR RFLP, DNA sequencing or, more recently DNA microsatellites, has improved the specific identification of larvae of morphologically non-differentiable Anisakis species, such as those of the A. simplex (s.l.) complex [12][13][14]. To date, it has not been established if any other Anisakis species can cause anisakiosis in humans [8].…”