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Anisakis pegreffii and A. simplex (s.s.) are the two zoonotic anisakids infecting cetaceans as well as pelagic/demersal fish and squids. In European waters, A. pegreffii prevails in the Mediterranean Sea, while A. simplex (s.s.) in the NE Atlantic Ocean. Abiotic conditions likely play a significant role in shaping their geographical distribution. The Iberian Atlantic and Alboran Sea waters are sympatric areas of the two species. A total of 429 adults and L3 stage from both sympatric and allopatric areas were studied by a wide nuclear genotyping approach (including newly and previously found diagnostic single nucleotide polymorphisms (SNPs) at nuclear DNA (nDNA) and microsatellite DNA loci) and sequenced at mitochondrial DNA (mtDNA) cox 2. Admixture between the two species was detected in the sympatric areas studied by STRUCTURE Bayesian analysis; NEWHYBRIDS revealed different categories of hybridization between the two species, representing approximately 5%. A tendency for F1 female hybrids to interbreed with the parental species at the geographical distribution limits of both species was observed. This finding suggests that hybridization occurs when the two parental species significantly differ in abundance. Mitochondrial introgression of A. simplex (s.s.) in A. pegreffii from Mediterranean waters was also detected, likely as a result of past and/or paleo-introgression events. The high level of genetic differentiation between the two species and their backcrosses indicates that, despite current hybridization, reproductive isolation which maintains evolutionary boundaries between the two species, exists. Possible causes of hybridization phenomena are attempted, as well as their evolutionary and ecological implications, also considering a sea warming scenario in European waters.

Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (=A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Anisakid nematodes are widespread marine parasites with complex life cycles involving invertebrates and fish as intermediate or transport hosts, and marine mammals as definitive hosts. Despite their ecological importance, and the zoonotic potential associated with the larval stages found in fish, recent data on anisakid species diversity in pinnipeds from Norwegian waters remain scarce. In this study, we investigated anisakid infections in two juvenile harbour seals ( Phoca vitulina ) stranded along the southern coast of Norway. Gastrointestinal nematodes were collected, morphologically classified to the genus level, and subsequently identified to species level through molecular analyses of mitochondrial (mtDNA cox2 ) and nuclear (rDNA ITS) markers. Five anisakid species were identified: Contracaecum osculatum sp. A (reported here for the first time in harbour seals), C. osculatum (sensu stricto), Phocanema decipiens (s.s.), P. krabbei , and Anisakis simplex (s.s.). The latter species was found in unexpectedly high abundance and in fully developed adult stages in one of the seals. Notably, these adult A. simplex (s.s.) exhibited large body size, in contrast with previous studies reporting either absence or minimal presence of adults in harbour seals. The underlying mechanisms promoting growth and reproductive development of A. simplex (s.s.) in this host species remain unclear, but may involve a combination of host-specific physiological traits, environmental factors, and parasite phenotypic plasticity. Gross pathological examination revealed multiple gastric and intestinal ulcers in the same seal, including seven crateriform lesions consistent with ulcerative gastritis and enteritis, associated with nematode attachment and feeding. These findings expand the current knowledge on anisakid diversity in P. vitulina and provide novel evidence of its role as a definitive host for A. simplex (s.s.) in Norwegian coastal waters. Furthermore, the results suggest that competitive interactions among anisakid species, combined with ecological and physiological host factors, may facilitate the development and maturation of A. simplex (s.s.) in harbour seals. Further studies are warranted to assess the frequency and health implications of such infections in wild pinniped populations.

Anisakid nematodes are the most infamous parasites occurring in seafood with ability to infect humans. In the present study, the infective stages of five anisakid larval types, including Anisakis types I and III, Terranova types I and II and Contracaecum larval type, as well as adult Anisakis paggiae are reported from 16 host species from New Caledonian waters. The specific identity of the larval types was investigated using ITS sequence data. Anisakis larval types I and III were identified as Anisakis typica and Anisakis brevispiculata, respectively, based on identical ITS sequences. However, the specific identity of the Terranova larval types and Contracaecum larval type remains unknown until a matching ITS sequence from a well-identified adult is available. Several fish host species are reported for the first time for anisakid larval types found in this study. Considering that third-stage larvae of anisakids are known to be the infective stage of the parasite for humans and the popularity of seafood in New Caledonia, presence of these parasites in New Caledonian fish is of high importance in terms of public health and raising awareness among various stakeholders. Although adult nematodes in the present study were identified as Anisakis paggiae, the spicule length is shorter in our specimens and falls within the range reported for Anisakis oceanicus previously reported in Pacific waters from black fish (genus Globicephala) and later synonymised with Anisakis physeteris. However, our specimens are different from A. physeteris in morphology of ventriculus. Anisakis paggiae has been reported from whales in southern hemisphere and this is the first report from the Pacific regions.

Parasites of the nematode genus Anisakis are associated with aquatic organisms. They can be found in a variety of marine hosts including whales, crustaceans, fish and cephalopods and are known to be the cause of the zoonotic disease anisakiasis, a painful inflammation of the gastro-intestinal tract caused by the accidental consumptions of infectious larvae raw or semi-raw fishery products. Since the demand on fish as dietary protein source and the export rates of seafood products in general is rapidly increasing worldwide, the knowledge about the distribution of potential foodborne human pathogens in seafood is of major significance for human health. Studies have provided evidence that a few Anisakis species can cause clinical symptoms in humans. The aim of our study was to interpolate the species range for every described Anisakis species on the basis of the existing occurrence data. We used sequence data of 373 Anisakis larvae from 30 different hosts worldwide and previously published molecular data (n = 584) from 53 field-specific publications to model the species range of Anisakis spp., using a interpolation method that combines aspects of the alpha hull interpolation algorithm as well as the conditional interpolation approach. The results of our approach strongly indicate the existence of species-specific distribution patterns of Anisakis spp. within different climate zones and oceans that are in principle congruent with those of their respective final hosts. Our results support preceding studies that propose anisakid nematodes as useful biological indicators for their final host distribution and abundance as they closely follow the trophic relationships among their successive hosts. The modeling might although be helpful for predicting the likelihood of infection in order to reduce the risk of anisakiasis cases in a given area.

Anisakid nematodes are a globally distributed group of marine mammal parasites. Kogiid whales, including the pygmy sperm whale Kogia breviceps, host an assemblage of specific anisakid species. Currently, three species are known to be specific to kogiid hosts, i.e., Skrjabinisakis paggiae, S. brevispiculata, and the less studied Pseudoterranova ceticola. The aim of this study was to investigate the species diversity of anisakid nematodes sampled from a pygmy sperm whale stranded in 2013 at the edge of its distribution range in the Northeast Atlantic, specifically in the North of Scotland. Nematodes were assigned to genus level based on morphology and identified by sequence analysis of the mtDNA cox2 gene and the rDNA ITS region. The present finding represents the first observation of syntopic occurrence of adult stages of S. brevispiculata, S. paggiae, and P. ceticola in a pygmy sperm whale in the Northeast Atlantic, and represent the northernmost record of these species in this area. Skrjabinisakis brevispiculata was the most abundant species, accounting for 55% of the identified nematodes, predominantly in the adult stage. Anisakis simplex (s.s.) was also abundant, with most specimens in the preadult stage, followed by S. paggiae and P. ceticola. The pygmy sperm whale is rarely documented in Scottish waters, and its occurrence in the area could suggest expansion of its geographic range. The presence of S. brevispiculata, S. paggiae, and P. ceticola in this whale species in this region may indicate a shift in the whole host community involved in the life cycle of these parasites in northern waters. However, it is also plausible that these parasites were acquired while the whale was feeding in more southern regions, before migrating northbound.

The East Asian finless porpoise, Neophocaena asiaeorientalis sunameri , is an endangered species that inhabits the coastal marine environments of East Asia. In the present study, we investigated the overall infection status of anisakid nematodes in East Asian finless porpoises from three sea sectors off the Korean Peninsula. The genetic diversity and population genetic structure of the identified nematode species were evaluated. The prevalence of all stages of anisakid nematodes collected from the stomach was 57.55% (61 among the 106 porpoises examined), and 16 of the hosts were found to have adult worms. The mean number of infected adults was 211 (± 419.54, 5–1455 per host). Only one species of anisakids, Anisakis pegreffii , was identified from randomly selected worms by molecular approaches. Analysis of the mitochondrial (mt) cox2 partial gene in 50 newly generated sequences of A. pegreffii revealed 24 haplotypes, including 14 new haplotypes. We observed below-average levels of nucleotide diversity and haplotype diversity compared to other seas around the world. The mtDNA cox2 haplotypes of the species in the three Korean sea areas showed no genetic structure, suggesting well-connected gene flow within these areas. This study represents the first record of a definitive host of A. pegreffii in Korean waters, providing important information regarding anisakids genetic diversity in the cetacean species inhabiting limited regions.

Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

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 EF1 α -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. Le genre Anisakis représente l’un des groupes de nématodes ascaridoïdes les plus répandus dans l’écosystème marin. Trois taxons étroitement apparentés sont reconnus dans le complexe Anisakis simplex (s. l.) : A. pegreffii , A. simplex (s. s.) et A. berlandi . Ils sont largement répartis dans les populations de leurs hôtes intermédiaires/paraténiques (poissons et calmars) et définitifs (cétacés). Un nouveau locus de gène nucléaire, la métallopeptidase 10 ( nas 10 ) (451 pb), a été séquencé et validé sur un total de 219 spécimens des trois espèces d’ Anisakis , collectés chez des hôtes poissons et cétacés de zones allopatriques incluses dans leur aire de répartition. Les échantillons d’ Anisakis ont d’abord été identifiés par des allozymes et une analyse des séquences de l’ADNmt cox2 et de l’ADNn EF1α-1. Le nouveau marqueur nucléaire a montré des positions de nucléotides alternatives fixes dans les trois espèces, c’est-à-dire qu’il a permis un diagnostic à 100%, permettant la détermination de l’espèce d’un grand nombre d’échantillons analysés dans la présente étude. De plus, des amorces à utiliser pour la PCR par système de mutation réfractaire à l’amplification (ARMS) du même locus génique ont été conçues à ces positions nucléotidiques. Ainsi, la détermination directe du génotypage, par double ARMS, a été développée et validée sur 219 spécimens appartenant aux trois espèces. Une concordance complète a été observée entre les dosages ARMS PCR tétra-amorces et les résultats de séquençage direct obtenus pour le locus du gène nas 10 . Le nouveau marqueur de diagnostic nucléaire sera utile dans les travaux futurs d’une approche de génotypage multi-locus et également pour étudier les éventuels événements d’hybridation et/ou d’introgression se produisant entre les trois espèces dans des zones sympatriques.