Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

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.).

Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real-time PCR.

BACKGROUND: Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide. The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. RESULTS: The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (-4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. CONCLUSION: The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health. © 2020 Society of Chemical Industry.

Anisakis spp. in toothed and baleen whales from Japanese waters with notes on their potential role as biological tags.

In this study, Anisakis nematodes isolated from toothed and baleen whales from localities around Japan were molecularly (PCR-RFLP) identified. In Wakayama, common bottlenose dolphins (Tursiops truncatus) were infected with A. simplex sensu stricto (s.s.), A. typica and A. pegreffii, while A. typica was the only species found in pantropical spotted dolphin (Stenella attenuata) and striped dolphin (S. coeruleoalba). Offshore common minke whales (Balaenoptera acutorostrata) and sei whales (B. borealis) were almost exclusively infected with A. simplex s.s.. However, in common minke whales from two Hokkaido localities, mature worms mostly consisted of A. simplex s.s. in some individuals and of A. pegreffii in others, but immature worms were mainly A. simplex s.s.. Gross and histopathological examination on gastric mucosa attached by anisakids resulted in mild and superficial reactions by the two baleen whale species in contrast to severe inflammatory reaction associated with ulcer formations by common bottlenose dolphin. Host specificity and adaptability of Anisakis spp. in these baleen and toothed whales were discussed from the points of view of adult worm size, worm population and pathological reactions by hosts. Interestingly, most of the common minke whales predominantly harboring mature A. pegreffii adults belonged to the Yellow Sea - East China Sea stock (J stock), which migrates through the Sea of Japan, whereas most of those mainly parasitized by mature A. simplex s.s. adults were from the Okhotsk Sea - West Pacific stock (O stock), mostly inhabiting the Pacific side, suggesting that these sibling species may have utility as biological tags to differentiate whale stocks. These results represent the first definitive host records for A. pegreffi in the Northwestern Pacific Ocean.

Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release.

BACKGROUND: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince. RESULTS: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases. CONCLUSION: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry.

A new species of Contracaecum (Nematoda: Anisakidae) found parasitizing Nannopterum brasilianus (Suliformes: Phalacrocoracidae) and Hoplias argentinensis (Characiformes: Erythrinidae) in South America: morphological and molecular characterization of larval and adult stages.

Nematode species of the genus Contracaecum Railliet & Henry, 1912 have been reported around the world in many species of fish-eating birds and seals. Here, Contracaecum jorgei n. sp. is morphologically described using light and scanning electron microscopy for adults and fourth-stage larvae (L4) found in the bird Nannopterum brasilianus and third-stage larvae (L3) found in the freshwater fish Hoplias argentinensis, both from the province of Córdoba, Argentina. Additionally, sequences of cytochrome c oxidase subunit II were obtained from these specimens and molecular phylogenetic analysis was used to determine its relationships within the genus. The present species is distinguished from other species by the number and disposition of cephalic papillae; shape and size of the interlabia; length of the spicules; and number and arrangement of papillae in the posterior end of the male. Furthermore, in the molecular analyses, sequences obtained from adult L4 and L3 specimens of C. jorgei n. sp. were similar and grouped, forming an independent lineage, thus confirming it as a distinct species. Thus, morphological characteristics associated with molecular data support the proposal of a new species.

Quantitative Proteomics Comparison of Total Expressed Proteomes of Anisakis simplex Sensu Stricto, A. pegreffii, and Their Hybrid Genotype.

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes' biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.

Larval ascaridoid nematodes in horned and musky octopus (Eledone cirrhosa and E. moschata) and longfin inshore squid (Doryteuthis pealeii): Safety and quality implications for cephalopod products sold as fresh on the Italian market.

The aim of this study was to evaluate the occurrence, infection level and distribution of ascaridoid larvae in cephalopod products sold in Italy. Data on the species most commonly commercialized as whole and fresh on the Italian market were collected. After comparing commercial and literature data, Eledone spp., comprising E. cirrhosa and E. moschata (horned octopus and musky octopus, respectively) and Doryteuthis pealeii (longfin inshore squid) were selected, as they had been rarely investigated. Overall, 75 Eledone spp. caught in the Mediterranean Sea (FAO area 37) and 70 D. pealeii from the Northwest Atlantic Ocean (FAO area 21) were examined by visual inspection and artificial digestion (viscera and mantle separately). Parasites were submitted to morphological and molecular analysis. Prevalence (P), mean intensity (MI) and mean abundance (MA) were calculated. In D. pealeii, 2 nematode larvae molecularly identified as Anisakis simplex s.s. were found in the viscera and in the mantle of two specimens (P: 2.9% 95% CI: 0-6.8%; MI: 1; MA: 0.028). In Eledone spp. 9 nematode larvae molecularly attributed to Hysterothylacium spp. were found in the mantle of 5 specimens (P: 6.7% 95% CI: 1-12.3%; MI: 1.8; MA: 0.12). This is the first report of A. simplex s.s. in D. pealeii. Considering the zoonotic and allergenic potential of these larvae and their localization also in the edible part (mantle), a potential public health issue exists.

Anisakid nematode larvae in the liver of Atlantic cod Gadus morhua L. from West Greenland.

Anisakid nematode larvae occur frequently in the liver of Atlantic cod, but merely few infection data from cod in waters around Greenland exist. The present study reports the occurrence of third-stage anisakid larvae in the livers of 200 Atlantic cod caught on fishing grounds along the West coast of Greenland (fjord systems of Maniitsoq) in May, June, August and September 2017. Classical and molecular helminthological techniques were used to identify the nematodes. A total of 200 cod livers were examined, and 194 were infected with third-stage nematode larvae (overall prevalence of infection 97%) with a mean intensity of 10.3 (range between 1 and 44 parasites per fish). Prevalences recorded were 96% for Anisakis simplex (s.l.), 55% for Pseudoterranova decipiens (s.l.) and 8% for Contracaecum osculatum (s.l.). Sequencing the mtDNA cox2 from 8 out of 23 these latter larvae conferred these to C. osculatum sp. B. A clear seasonal variation was observed, with a rise in A. simplex (s.l.) and P. decipiens (s.l.) occurrence in June and August and a decline in September. The study may serve as a baseline for future investigations using the three anisakids as biological indicators in Greenland waters.

A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events.

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved ß-tubulin gene (ß-TUB). The two cryptic species differed at least in 7 bp in the ß-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and ß-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

First report of Anisakis sp. (Nematoda: Anisakidae) parasitizing Muscovy duck in Marajó Island, state of Pará, Brazil.

Among the avian used for human consumption, the Muscovy duck is well adapted to various climatic conditions and its breeding is widespread due to its easy handling, and its meat is widely consumed and appreciated, especially in the cuisine of northern Brazil. The present study aimed to report and identify taxonomically the nematodes found in the esophagus of Muscovy ducks reared and marketed in the municipality of Soure, Marajó Island, state of Pará, Brazil, and discuss its zoonotic potential to human. The samples consisted of 30 specimens of Cairina moschata domestica analyzed. A total of 258 nematodes were recovered, which were strongly fixed in the esophageal mucosa of hosts. The morphological and morphometric characteristics were compatible with Anisakis third-stage larvae.

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).

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.

TITLE: Un nouveau marqueur nucléaire et développement d'un test ARMS-PCR ciblant le locus de la métallopeptidase 10 (nas 10) pour identifier les espèces du complexe Anisakis simplex (s. l.) (Nematoda, Anisakidae). ABSTRACT: 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.

Anisakis and Hysterothylacium species in Mediterranean and North-East Atlantic fishes commonly consumed in Spain: Epidemiological, molecular and morphometric discriminant analysis.

The consumption of raw fish parasitized with larval ascaridoid nematodes of the family Anisakidae can cause anisakiasis, provoking gastrointestinal and/or allergic symptomatology. The main causative agents in the Anisakis genus are the sibling species Anisakis simplex sensu stricto (s.s.) and A. pegreffii of the A. simplex sensu lato (s.l.) complex. Larvae of A. simplex (s.l.) are frequently detected in fish commonly consumed in Spain, as are larvae of the genus Hysterothylacium of the family Raphidascarididae, associated with allergic reactions but not considered pathogenic. Reported here are the results of an epidemiological survey of ascaridoid larvae in three commonly consumed fish species in Spain, horse mackerel (Trachurus trachurus) (n = 52), blue whiting (Micromesistius poutassou) (n = 93) and anchovy (Engraulis encrasicolus) (n = 69), caught in the North-Eastern Atlantic, West Mediterranean and Adriatic Sea. The larvae found in the dissected fish were identified in the following order of abundance: A. simplex (s.l.) (n = 2003), Hysterothylacium aduncum (n = 422), H. fabri (n = 180) and A. physeteris (n = 15). Binomial regression analysis showed a correlation between A. simplex (s.l.) and Hysterothylacium larvae abundance and the host geographical location, the North-Eastern Atlantic being the area with the highest parasitation. Fish length and weight and Fulton's condition factor were correlated with A. simplex (s.l.) abundance only in horse mackerel. There was a significant presence of A. simplex (s.l.) and H. aduncum larvae in the musculature of North-Eastern Atlantic blue whiting, the most parasitized part being the anteroventral region, followed equally by the anterodorsal and central sections. The ITS rDNA of larvae of the sibling species A. simplex (s.s.) and A. pegreffii was identified by PCR-RFLP, and a binary logistic regression model was developed to study their morphometric differentiation. Anisakis simplex (s.s.) was detected in the North-Eastern Atlantic and A. pegreffii in all the areas studied. The morphometric analysis discriminated between the two species at the third and fourth larval stages (L3 and L4), the latter obtained by in vitro culture in RPMI-1640 medium. Two discriminant functions were obtained for the L3 and L4 larvae, the ventricle being a key parameter for specific differentiation in both stages, providing taxonomical criteria that could be used besides molecular identification. The present study reveals differences in the parasitation of the studied fish, including the distribution of larvae in the musculature, related to the host species and its geographical origin.

New records of anisakid nematodes from marine fishes off New Caledonia, with descriptions of five new species of Raphidascaris (Ichthyascaris) (Nematoda, Anisakidae).

Recent examinations of anisakid nematodes (Anisakidae) from marine fishes off New Caledonia, collected in the years 2003-2008, revealed the presence of the following five new species of Raphidascaris Railliet et Henry, 1915, all belonging to the subgenus Ichthyascaris Wu, 1949: Raphidascaris (Ichthyascaris) spinicauda n. sp. from the redbelly yellowtail fusilier Caesio cuning (Caesionidae, Perciformes); Raphidascaris (Ichthyascaris) fasciati n. sp. from the blacktip grouper Epinephelus fasciatus (Serranidae, Perciformes); Raphidascaris (Ichthyascaris) nudicauda n. sp. from the brushtooth lizardfish Saurida undosquamis (Synodontidae, Aulopiformes); Raphidascaris (Ichthyascaris) euani n. sp. from the Japanese large-eye bream Gymnocranius euanus (Lethrinidae, Perciformes); and Raphidascaris (Ichthyascaris) elopsis n. sp. from the Hawaiian ladyfish Elops hawaiensis (Elopidae, Elopiformes). An additional two congeneric species, R. (I.) etelidis Moravec et Justine, 2012 and R. (I.) sillagoides (Bruce, 1990) were found in the deep-water red snapper Etelis carbunculus (new host record) and the deepwater longtail red snapper Etelis coruscans (both Lutjanidae, Perciformes), and the silver sillago Sillago sihama (Sillaginidae, Perciformes) (new host and geographical records), respectively. Two unidentified congeneric species, Raphidascaris (Ichthyascaris) sp. 1 from the trumpet emperor Lethrinus miniatus (Lethrinidae, Perciformes) and Raphidascaris (Ichthyascaris) sp. 2 from the white-spotted puffer Arothron hispidus (Tetraodontidae, Tetraodontiformes) were recorded. Moreover, two species of Hysterothylacium Ward et Magath, 1917, H. alatum Moravec et Justine, 2015 and H. epinepheli (Yamaguti, 1941), were found in the leopard coralgrouper Plectropomus leopardus (type host) and the highfin grouper Epinephelus maculatus (new host) (both Serranidae, Perciformes), respectively. This is the second finding of H. epinepheli since its original description in Japan 79 years ago. Most species are described based on light and electron microscopical studies.

TITLE: Nouvelles mentions de nématodes anisakidés de poissons marins de Nouvelle-Calédonie, avec description de cinq nouvelles espèces de Raphidascaris (Ichthyascaris) (Nematoda, Anisakidae). ABSTRACT: L'examen récent de nématodes Anisakidae de poissons marins de la Nouvelle-Calédonie, collectés dans les années 2003­2008, a révélé la présence des cinq nouvelles espèces de Raphidascaris Railliet et Henry, 1915, toutes appartenant au sous-genre Ichthyascaris Wu, 1949 : Raphidascaris (Ichthyascaris) spinicauda n. sp. chez le fusilier Caesio cuning (Caesionidae, Perciformes) ; Raphidascaris (Ichthyascaris) fasciati n. sp. chez la loche Epinephelus fasciatus (Serranidae, Perciformes) ; Raphidascaris (Ichthyascaris) nudicauda n. sp. chez le poisson-lézard Saurida undosquamis (Synodontidae, Aulopiformes) ; Raphidascaris (Ichthyascaris) euani n. sp. chez le bossu Gymnocranius euanus (Lethrinidae, Perciformes) ; et Raphidascaris (Ichthyascaris) elopsis n. sp. chez Elops hawaiensis (Elopidae, Elopiformes). Deux autres espèces congénériques, R. (I.) etelidis Moravec et Justine, 2012 et R. (I.) sillagoides (Bruce, 1990) ont été trouvées respectivement chez les vivaneaux de profondeur Etelis carbunculus (nouvel hôte) et Etelis coruscans (Lutjanidae, Perciformes) et chez Sillago sihama (Sillaginidae, Perciformes) (nouvel hôte et nouvelle mention géographique). Deux espèces congénériques non identifiées, Raphidascaris (Ichthyascaris) sp. 1 chez le bossu Lethrinus miniatus (Lethrinidae, Perciformes) et Raphidascaris (Ichthyascaris) sp. 2 chez Arothron hispidus (Tetraodontidae, Tetraodontiformes) sont signalées. De plus, deux espèces d'Hysterothylacium Ward et Magath, 1917, H. alatum Moravec et Justine, 2015 et H. epinepheli (Yamaguti, 1941), ont été trouvées chez la saumonée Plectropomus leopardus (hôte-type) et chez la loche uitoé Epinephelus maculatus (nouvel hôte) (Serranidae, Perciformes), respectivement. Il s'agit de la deuxième mention d'H. epinepheli depuis sa description originale au Japon il y a 79 ans. La plupart des espèces ont été décrites sur la base d'études au microscope optique et électronique.

Species of Anisakidae nematodes and Clinostomum spp. infecting lisa Mugil curema (Mugilidae) intended for human consumption in Mexico.

Anisakisspp. nematodes are potentially zoonotic parasites; that infects a wide variety of aquatic species worldwide, with marine fish being the paratenic hosts. The aim of study was identify the presence of Anisakidae nematodes, and other parasites in Mugil curema . A total of 96 M . curema obtained from local markets in Tulancingo, Hidalgo, Mexico, were analyzed by necropsy. Only five M . curema present nematode collection in epaxial muscle. The tissues with the highest prevalence of parasites were identified, and samples of epaxial muscle with larval migration analyzed by histopathology. Visible parasites in necropsy tissues were classified according to their morphology. Nematode found in the liver were Contracaecum spp. (41.17%) and Pseudoterranova spp. third stage (7.36%); in the caudal part of the kidney were Anisakis spp. (13.23%), Pseudoterranova spp. third stage (11.77%) and Contracaecum spp. (5.88%); and in epaxial muscle were Anisakis spp. Larva I (5.88%) and Pseudoterranova spp (4.42%). In one fish, Clinostomum spp. was detected in epaxial caudal muscle. The present work reports for the first time the presence of nematodes of the family Anisakidae and Clinostonum spp. metacercariae, with zoonotic potential, in M . curema intended for human consumption in Tulancingo, Hidalgo, Mexico.

Comparative Transcriptomics Reveals Clues for Differences in Pathogenicity between Hysterothylacium aduncum, Anisakissimplex sensu stricto and Anisakis pegreffii.

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.

Risk-based scoring and genetic identification for anisakids in frozen fish products from Atlantic FAO areas.

BACKGROUND: The presence of Anisakis larvae in fish represents a major public health concern. Effective risk management procedures should be applied to prevent heavily infected products from reaching the market. The aim of the study is to provide preliminary data on parasite exposure and risk classification in frozen fish products by applying a risk categorization scheme (site, abundance, density and epidemiology - SADE) and Fish Parasite Rating (FPR) method. Fish and cephalopods samples (N = 771) from 5 different FAO Atlantic areas were examined and categorized after an accurate visual inspection and a chloro-peptic digestion. RESULTS: In 25 out of 33 fish species parasite larvae were found. 10897 anisakids larvae were collected and identified to genus level. Molva dypterygia, Conger conger, Zeus faber and Aphanopus carbo were shown to be the most highly infected species. SADE and FPR scores were 1 and poor, respectively, for the referred species, because of the disseminated Anisakis infection and commercial rejection. CONCLUSION: SADE/FPR method showed high specificity and accuracy. The information provided in this work could be used in early warning systems for the detection of parasites in fishery products and might help fishing industries in establishing management strategies for infected stocks in terms of cost saving decisions.

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.

Multidisciplinary approach in study of the zoonotic Anisakis larval infection in the blue mackerel (Scomber australasicus) and the largehead hairtail (Trichiurus lepturus) in Northern Taiwan.

BACKGROUND: Anisakid larvae are the food-borne pathogen highly prevalent among numerous marine fishes. Accidental consumption of infected raw or poorly cooked fish fillets may cause anisakiasis. METHODS: This study used the multidisciplinary approach to investigate the occurrence of Anisakis nematodes in commonly consumed fish species, Scomber australasicus and Trichiurus lepturus purchased in Taipei Xinyi traditional fish market. RESULTS: All the Anisakis larvae collected herein were identified morphologically as Anisakis type I or Anisakis type II. The prevalence and the mean intensity of Anisakis larvae collected from S. australasicus was 80.77%, 26.8 (10-32) and 100%, 49.0 (27-70) for T. lepturus. Using molecular analysis, 83.33% (180/216) were identified as Anisakis pegreffii, 6.05% (13/216) as Ascaris typica, 1.85% (4/216) as Ascaris physeteris and 8.80% (19/216) as hybrid genotype (A. pegreffii + Anisakis simplex) in S. australasicus. In T. lepturus, 86.31% (290/336) were identified as A. pegreffii, 2.38% (8/336) as A. typica, and 11.31% (38/336) as hybrid genotype (A. pegreffii + A. simplex [s.s]). The molecular phylogenetic analysis shows two cluster clades, one group includes A. pegreffii complex and the other include Ascaris paggiae, Ascaris brevispiculata, and A. physeteris. CONCLUSION: Thus, A. pegreffii is the most abundant species and may be the potential causes of human infection.

Species of Anisakidae nematodes and Clinostomum spp. infecting lisa Mugil curema (Mugilidae) intended for human consumption in Mexico / Espécies de nematoides Anisakidae e Clinostomum spp. infectando tainhas Mugil curema (Mugilidae) destinadas ao consumo humano no México

Abstract Anisakisspp. nematodes are potentially zoonotic parasites; that infects a wide variety of aquatic species worldwide, with marine fish being the paratenic hosts. The aim of study was identify the presence of Anisakidae nematodes, and other parasites in Mugil curema . A total of 96 M . curema obtained from local markets in Tulancingo, Hidalgo, Mexico, were analyzed by necropsy. Only five M . curema present nematode collection in epaxial muscle. The tissues with the highest prevalence of parasites were identified, and samples of epaxial muscle with larval migration analyzed by histopathology. Visible parasites in necropsy tissues were classified according to their morphology. Nematode found in the liver were Contracaecum spp. (41.17%) and Pseudoterranova spp. third stage (7.36%); in the caudal part of the kidney were Anisakis spp. (13.23%), Pseudoterranova spp. third stage (11.77%) and Contracaecum spp. (5.88%); and in epaxial muscle were Anisakis spp. Larva I (5.88%) and Pseudoterranova spp (4.42%). In one fish, Clinostomum spp. was detected in epaxial caudal muscle. The present work reports for the first time the presence of nematodes of the family Anisakidae and Clinostonum spp. metacercariae, with zoonotic potential, in M . curema intended for human consumption in Tulancingo, Hidalgo, Mexico.

Resumo Anisakisspp. são parasitas potencialmente zoonóticos que infectam uma grande variedade de espécies aquáticas em todo o mundo, sendo os peixes marinhos hospedeiros paratênicos. O objetivo deste estudo foi identificar a presença de nematóides da família Anisakidae e de outros parasitas em peixes Mugil curema . Um total de 96 M . curema , obtidos em mercados locais em Tulancingo, Hidalgo, México, foram submetidos a necropsia. Apenas cinco M. curema apresentaram coleção de nematóides no músculo epaxial. Os tecidos com maior prevalência de parasitas foram identificados e amostras do músculo epaxial com migração larval foram analisadas por histopatologia. Os nematóides encontrados no fígado foram Contracaecum spp. (41,17%) e Pseudoterranova spp. terceira etapa (7,36%); na parte caudal do rim Anisakis spp. (13,23%), Pseudoterranova spp. terceira etapa (11,77%) e Contracaecum spp. (5,88%); e no músculo epaxial Anisakis spp. larva I (5,88%) e Pseudoterranova spp. (4,42%). Em um peixe, Clinostomum spp. foi detectado no músculo caudal epaxial. O presente trabalho relata pela primeira vez a presença de nematóides da família Anisakidae e Clinostonum spp. metacercárias, com potencial zoonótico, em M. curema destinado ao consumo humano em Tulancingo, Hidalgo, México.