Investigating the genetic structure of the parasites Anisakis pegreffii and A. berlandi (Nematoda: Anisakidae) in a sympatric area of the southern Pacific Ocean waters using a multilocus genotyping approach: first evidence of their interspecific hybridization.

The southern Pacific Ocean, off the New Zealand coast, has been reported as one sympatric area of the two parasite species Anisakis pegreffii and A. berlandi. Here, a multilocus genotyping approach, based on a panel of eleven DNA microsatellite (SSR) loci plus the sequences analysis of the nuclear nas10 nDNA and the mitochondrial mtDNA cox2 gene loci, was applied to a total of N = 344 adults and larvae of Anisakis spp. from cetacean and fish species, respectively. Out of the newly scored SSR loci, Anisl 15 and Anisl 2 showed fixed alternative alleles between A. pegreffii and A. berlandi resulting as 100% diagnostic loci. Out of SSRs Anisl 00314 and Anisl 7 previously disclosed, two additional loci, i.e., Anisl 4 and Anisl 22, were found to be sex-linked. The Bayesian genotypes clustering approach (STRUCTURE) allowed identification, with a 100% of probability value, N = 208 specimens to the "pure parental" A. pegreffii, N = 133 to the "pure parental" A. berlandi, while one adult and two larval stages showed mixed ancestry between the two groups having, in all cases, a Q-value = 0.50. NEWHYBRIDS analysis assigned (100% of probability) those specimens to their F1 hybrid category. This represents the first evidence of contemporary hybridization between the two parasite species in a sympatric area. The pairwise FST values estimated at intraspecific and interspecific level, inferred from both SSR loci and mitochondrial mtDNA cox2 sequences, have also demonstrated the existence of two distinct panmictic units in this study area, corresponding respectively to A. pegreffii and A. berlandi. The results obtained support the useful application of a multilocus approach in the identification of sibling species and their hybrid categories in sympatric areas. The possible use of sex-linked SSR loci of the two species of the A. simplex (s. l.), for sex determination of their larval stages, is also suggested.

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

Eight microsatellite loci, recently developed in the species Anisakis pegreffii, were successfully amplified in Anisakis berlandi, sibling species of the A. simplex (s. l.) complex. They were validated on adult specimens (n = 46) of the parasite species, collected from two individuals of the definitive host, the long-finned pilot whale Globicephala melas from New Zealand waters. Among the eight loci scored, one, Anisl 07132, had null alleles in A. berlandi and was thus excluded from the subsequent genetic analysis. Two loci, Anisl 00314 and Anisl 10535, were monomorphic. In addition, as also previously detected in the other species of the A. simplex (s. l.) complex, the Anisl 7 locus was seen to be sex-linked, showing hemizygosity in male specimens. Differential allele frequency distributions of A. berlandi, with respect to those previously observed in A. pegreffii and A. simplex (s. s.), were found at some microsatellite loci. The Anisl 7 locus provided 100% diagnosis between A. berlandi and A. pegreffii, while others resulted in 99% diagnosis between A. berlandi and the other two species. Simple sequence repeat (SSR) loci also allowed us to estimate the genetic differentiation of A. berlandi from A. pegreffii (F st ≈ 0.45, Dc = 0.82) and A. simplex (s. s.) (F st ≈ 0.57, Dc = 0.73). The results suggest that SSRs provide a set of candidate markers for population genetics analysis of A. berlandi, as well as for the investigation, through a multi-locus genotyping approach, of possible patterns of hybridisation/introgression events between A. berlandi and the other two Anisakis species in sympatric conditions.

TITLE: Utilité des loci microsatellites pour la caractérisation génétique interspécifique d'Anisakis berlandi (Nematoda, Anisakidae). ABSTRACT: Huit loci microsatellites, récemment développés chez l'espèce Anisakis pegreffii, ont été amplifiés avec succès chez Anisakis berlandi, espèce sœur du complexe A. simplex (s. l.). Ils ont été validés sur des spécimens adultes (n = 46) de l'espèce, récoltés chez deux individus de l'hôte définitif, le globicéphale commun Globicephala melas, des eaux néo-zélandaises. Parmi les huit loci notés, l'un, Anisl 07132, avait des allèles nuls chez A. berlandi et a donc été exclu de l'analyse génétique ultérieure. Deux loci, Anisl 00314 et Anisl 10535, étaient monomorphes. De plus, comme cela a également été détecté précédemment dans les autres espèces du complexe A. simplex (s. l.), le locus Anisl 7 était lié au sexe, montrant une hémizygosité chez les spécimens mâles. Chez A. berlandi, des distributions de fréquences d'allèles, différentielles par rapport à celles précédemment observées chez A. pegreffii et A. simplex (s. s.), ont été trouvées pour certains loci microsatellites. Le locus Anisl 7 a fourni un diagnostic à 100 % entre A. berlandi et A. pegreffii, tandis que d'autres ont abouti à un diagnostic à 99 % entre A. berlandi et les deux autres espèces. Les loci des SSR ont également permis d'estimer la différenciation génétique d'A. berlandi par rapport à A. pegreffii (F st ≈ 0,45, Dc = 0,82) et A. simplex (s. s.) (F st ≈ 0,57, Dc = 0,73). Les résultats suggèrent que les répétitions de séquences simples (SSR) fournissent un ensemble de marqueurs candidats pour l'analyse génétique des populations d'A. berlandi, ainsi que pour l'investigation, dans une approche de génotypage multilocus, des modèles possibles d'hybridation/introgression entre A. berlandi et les deux autres espèces d'Anisakis dans des conditions sympatriques.

Novel polymorphic microsatellite loci in Anisakis pegreffii and A. simplex (s. s.) (Nematoda: Anisakidae): implications for species recognition and population genetic analysis.

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.