Distribution and genetic diversity of Anisakis spp. in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea.

Parasite biodiversity in cetaceans represents a neglected component of the marine ecosystem. This study aimed to investigate the distribution and genetic diversity of anisakid nematodes of the genus Anisakis sampled in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea. A total of 478 adults and pre-adults of Anisakis spp. was identified by a multilocus genetic approach (mtDNA cox2, EF1 α - 1 nDNA and nas 10 nDNA gene loci) from 11 cetacean species. A clear pattern of host preference was observed for Anisakis spp. at cetacean family level: A. simplex (s.s.) and A. pegreffii infected mainly delphinids; A. physeteris and A. brevispiculata were present only in physeterids, and A. ziphidarum occurred in ziphiids. The role of cetacean host populations from different waters in shaping the population genetic structure of A. simplex (s.s.), A. pegreffii and A. physeteris was investigated for the first time. Significant genetic sub-structuring was found in A. simplex (s.s.) populations of the Norwegian Sea and the North Sea compared to those of the Iberian Atlantic, as well as in A. pegreffii populations of the Adriatic and the Tyrrhenian Seas compared to those of the Iberian Atlantic waters. Substantial genetic homogeneity was detected in the Mediterranean Sea population of A. physeteris. This study highlights a strong preference by some Anisakis spp. for certain cetacean species or families. Information about anisakid biodiversity in their cetacean definitive hosts, which are apex predators of marine ecosystems, acquires particular importance for conservation measures in the context of global climate change phenomena.

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.

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.

Genetic identification and insights into the ecology of Contracaecum rudolphii A and C. rudolphii B (Nematoda: Anisakidae) from cormorants and fish of aquatic ecosystems of Central Italy.

Contracaecum rudolphii (s. l.) is a complex of sibling species of anisakid nematodes having the fish-eating birds belonging to the Family Phalacrocoracidae as final hosts. The great cormorant Phalacrocorax carbo sinensis is parasitized by C. rudolphii A and C. rudolphii B. Adults and L4 specimens of C. rudolphii (s. l.) (N = 3282) were collected in cormorants from brackish and freshwater ecosystems of Central Italy. Third-stage larvae of Contracaecum (N = 882) were obtained from the fish species Dicentrarchus labrax, Anguilla anguilla, Aphanius fasciatus, Atherina boyeri, Leuciscus cephalus, Barbus barbus, and Carassius carassius captured in the same geographical areas of cormorants' standings. Contracaecum rudolphii A and C. rudolphii B were identified by a multilocus genetic approach: allozymes, sequences analysis of the mtDNA cox2, and ITS region of rDNA gene loci. Differential distribution of the two parasite species was observed in different aquatic environments. Contracaecum rudolphii B outnumbered C. rudolphii A in wintering cormorants from freshwater ecosystems; the opposite trend was found in cormorants from brackish water. Analogously, C. rudolphii A larvae were more prevalent in brackish water fish, while C. rudolphii B larvae were found infecting only freshwater fish. The findings seem to confirm that C. rudolphii A and C. rudolphii B would have a life-cycle adapted to brackish and freshwater environments, respectively. A differential feeding behavior of wintering cormorants, the ecology of the infected fish species, and abiotic factors related to early stages of the parasites are supposed to maintain the distinctiveness of the two parasite species' life cycles in the two different aquatic ecosystems.

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.

Gene expression profiles of antigenic proteins of third stage larvae of the zoonotic nematode Anisakis pegreffii in response to temperature conditions.

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host's immune response.

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.

Morphological and molecular identification of a new Kudoa thyrsites isolate in Mediterranean silver scabbardfish Lepidopus caudatus.

Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host's musculature, or cause post mortem myoliquefaction, commonly known as 'soft flesh'. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Mediterranean Sea). For comparative and validation purposes, the morphological and molecular characteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stellate appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously recorded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characterization of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores.

Molecular Epidemiology of Anisakis and Anisakiasis: An Ecological and Evolutionary Road Map.

This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.

Expanded description of Lamproglena cleopatra Humes, 1957 (Lernaeidae: Copepoda) from Labeo spp. (Cyprinidae) with a key to species of Lamproglena von Nordmann, 1832.

The occurrence of the copepod Lamproglena cleopatra Humes, 1957, parasitising freshwater fishes in the Limpopo River System is presented, along with new morphological data. This crustacean was originally described parasitising a cyprinid (Labeo forskalii Rüppell) from the River Nile, Egypt. During 2014-2015 crustacean samples were collected from the gills of three cyprinid fish species, Labeo rosae Steindachner from Flag Boshielo Dam, Labeo molybdinus Du Plessis from Nwanedi-Luphephe Dam in South Africa, and Labeo ruddi Boulenger from the River Bubye in Zimbabwe. The specimens from the present study were morphologically similar regardless of the host, but exhibited some morphometric intraspecific differences in comparison with the type-specimen from Egypt. A description of L. cleopatra copepodid III stage and a taxonomic key to Lamproglena spp. is provided.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay.

BACKGROUND: Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. METHODS: Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied. RESULTS: According to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like. CONCLUSION: The Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

Species composition and infection dynamics of ascaridoid nematodes in Barents Sea capelin (Mallotus villosus) reflecting trophic position of fish host.

Capelin (Mallotus villosus) is among the most abundant fish species in the Barents Sea, and represents a critical food source for many predators in the area including Atlantic cod and harp seal. In Norway, the fish is of economic importance since whole capelin and roe are valuable export products. Despite its economic and ecological importance, the parasites of Barents Sea capelin are poorly known. However, the presence of parasites in the edible parts may adversely affect product quality and consumer safety. During the main annual catching seasons of 2009-2012, we investigated the diversity and infection dynamics of ascaridoid nematodes in capelin (n = 620) from the southern Barents Sea. Three anisakid species were identified by genetic or molecular methods; Anisakis simplex (s.s.), Contracaecum osculatum sp. B, and Hysterothylacium aduncum, with C. osculatum sp. B as the most prevalent and abundant species. The present findings suggest that the ascaridoid species composition in capelin reflects its trophic position in the Barents Sea ecosystem. There appears to be a link between infection level of the nematode species and the preferred prey organisms of the different developmental phases of capelin. Thus, the higher abundance of C. osculatum sp. B compared to A. simplex (s.s.) and H. aduncum may be related to more extensive feeding on calanoid copepods over a wider ontogenetic size range including adolescence, while the main intermediate hosts of the latter nematode species, i.e. euphausiids and amphipods, appear to be the preferred prey of larger capelin.

DNA-Based Taxonomy in Ecologically Versatile Microalgae: A Re-Evaluation of the Species Concept within the Coccoid Green Algal Genus Coccomyxa (Trebouxiophyceae, Chlorophyta).

Coccomyxa is a genus of unicellular green algae of the class Trebouxiophyceae, well known for its cosmopolitan distribution and great ecological amplitude. The taxonomy of this genus has long been problematic, due to reliance on badly-defined and environmentally variable morphological characters. In this study, based on the discovery of a new species from an extreme habitat, we reassess species circumscription in Coccomyxa, a unicellular genus of the class Trebouxiophyceae, using a combination of ecological and DNA sequence data (analyzed with three different methods of algorithmic species delineation). Our results are compared with those of a recent integrative study of Darienko and colleagues that reassessed the taxonomy of Coccomyxa, recognizing 7 species in the genus. Expanding the dataset from 43 to 61 sequences (SSU + ITS rDNA) resulted in a different delimitation, supporting the recognition of a higher number of species (24 to 27 depending on the analysis used, with the 27-species scenario receiving the strongest support). Among these, C. melkonianii sp. nov. is described from material isolated from a river highly polluted by heavy metals (Rio Irvi, Sardinia, Italy). Analyses performed on ecological characters detected a significant phylogenetic signal in six different characters. We conclude that the 27-species scenario is presently the most realistic for Coccomyxa and we suggest that well-supported lineages distinguishable by ecological preferences should be recognized as different species in this genus. We also recommend that for microbial lineages in which the overall diversity is unknown and taxon sampling is sparse, as is often the case for green microalgae, the results of analyses for algorithmic DNA-based species delimitation should be interpreted with extreme caution.

Temporal stability of parasite distribution and genetic variability values of Contracaecum osculatum sp. D and C. osculatum sp. E (Nematoda: Anisakidae) from fish of the Ross Sea (Antarctica).

The Ross Sea, Eastern Antarctica, is considered a "pristine ecosystem" and a biodiversity "hotspot" scarcely impacted by humans. The sibling species Contracaecum osculatum sp. D and C. osculatum sp. E are anisakid parasites embedded in the natural Antarctic marine ecosystem. Aims of this study were to: identify the larvae of C. osculatum (s.l.) recovered in fish hosts during the XXVII Italian Expedition to Antarctica (2011-2012); perform a comparative analysis of the contemporary parasitic load and genetic variability estimates of C. osculatum sp. D and C. osculatum sp. E with respect to samples collected during the expedition of 1993-1994; to provide ecological data on these parasites. 200 fish specimens (Chionodraco hamatus, Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi) were analysed for Contracaecum sp. larvae, identified at species level by allozyme diagnostic markers and sequences analysis of the mtDNA cox2 gene. Statistically significant differences were found between the occurrence of C. osculatum sp. D and C. osculatum sp. E in different fish species. C. osculatum sp. E was more prevalent in T. bernacchii; while, a higher percentage of C. osculatum sp. D occurred in Ch. hamatus and T. hansoni. The two species also showed differences in the host infection site: C. osculatum sp. D showed higher percentage of infection in the fish liver. High genetic variability values at both nuclear and mitochondrial level were found in the two species in both sampling periods. The parasitic infection levels by C. osculatum sp. D and sp. E and their estimates of genetic variability showed no statistically significant variation over a temporal scale (2012 versus 1994). This suggests that the low habitat disturbance of the Antarctic region permits the maintenance of stable ecosystem trophic webs, which contributes to the maintenance of a large populations of anisakid nematodes with high genetic variability.

Genetic and morphological approaches distinguish the three sibling species of the Anisakis simplex species complex, with a species designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae).

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.

Molecular identification, morphological characterization and new insights into the ecology of larval Pseudoterranova cattani in fishes from the Argentine coast with its differentiation from the Antarctic species, P. decipiens sp. E (Nematoda: Anisakidae).

Larvae of the genus Pseudoterranova constitute a risk for human health when ingested through raw or undercooked fish. They can provoke pseudoterranovosis in humans, a fish-borne zoonotic disease whose pathogenicity varies with the species involved, making their correct specific identification a necessary step in the knowledge of this zoonosis. Larvae of Pseudoterranova decipiens s.l. have been reported in several fish species from off the Argentine coasts; however, there are no studies dealing with their specific identification in this region. Here, a genetic identification and morphological characterization of larval Pseudoterranova spp. from three fish species sampled from Argentine waters and from Notothenia coriiceps from Antarctic waters was carried out. Larvae were sequenced for their genetic/molecular identification, including the mitochondrial cytochrome c oxidase subunit II (mtDNA cox2), the first (ITS-1) and the second (ITS-2) internal transcribed spacers of the nuclear ribosomal DNA, and compared with all species of the P. decipiens (sensu lato) species complex (sequences available in GenBank). Further, adults of Pseudoterranova spp. from the definitive host, the southern sea lion, Otaria flavescens, from Argentine and Chilean coasts were sequenced at the same genes. The sequences obtained at the ITS-1 and ITS-2 genes from all the larvae examined from fish of Argentine waters, as well as the adult worms, matched 100% the sequences for the species P. cattani. The sequences obtained at mtDNA cox2 gene for Antarctic larvae matched 99% those available in GenBank for the sibling P. decipiens sp. E. Both MP and BI phylogenetic trees strongly supported P. cattani and P. decipiens sp. E as two distinct phylogenetic lineages and depicted the species P. decipiens sp. E as sister taxon to the remaining taxa of the P. decipiens complex. Larval morphometry was similar between specimens of P. cattani from Argentina, but significantly different from those of P. decipiens sp. E, indicating that larval forms can be distinguished based on their morphology. Pseudoterranova cattani is common and abundant in a variety of fish species from Chile, whereas few host species harbour these larvae in Argentina where they show low levels of parasitism. This pattern could arise from a combination of factors, including environmental conditions, density and dietary preferences of definitive hosts and life-cycle pathways of the parasite. Finally, this study revealed that the life-cycle of P. cattani involves mainly demersal and benthic organisms, with a marked preference by large-sized benthophagous fish.

Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy.

Human cases of gastric anisakiasis caused by the zoonotic parasite Anisakis pegreffii are increasing in Italy. The disease is caused by ingestion of larval nematodes in lightly cooked or raw seafood. Because symptoms are vague and serodiagnosis is difficult, the disease is often misdiagnosed and cases are understimated.

Molecular identification and larval morphological description of Contracaecum pelagicum (Nematoda: Anisakidae) from the anchovy Engraulis anchoita (Engraulidae) and fish-eating birds from the Argentine North Patagonian Sea.

Anisakids use invertebrates as paratenic and/or intermediate hosts as a basic feature of larval transmission. The third-stage larva usually develops in invertebrates which are prey items of finfish paratenic hosts. Contracaecum larvae molt twice inside the egg and hatch as free third-stage larvae ensheathed in the second-stage larval cuticle. Copepods act as paratenic or obligatory hosts, usually ingesting these free L3 larvae, and fish act as intermediate/paratenic or metaparatenic hosts preying on infected copepods. Fish-eating birds acquire L3 larvae by ingesting infected fish where they develop into the fourth-stage larvae and adults. Objectives of this work were to establish the specific correspondence between Contracaecum pelagicum L3 larvae parasitizing the anchovy Engraulis anchoita, and the adults parasitizing the Magellanic penguin Spheniscus magellanicus and the Imperial shag Phalacrocorax atriceps through the use of molecular markers; and, to evaluate the anisakid L3 larval recruitment and infection caused by ingestion of anchovy by S. magellanicus. Sixteen specimens of Contracaecum L3 larvae were analyzed from E. anchoita from Bahía Engaño, Chubut, eight adult nematodes from S. magellanicus and six adult specimens from P. atriceps both from the Valdés Peninsula, Chubut. All nematodes were sequenced for three genes: mitochondrial cytochrome oxidase 2 (mtDNA cox2), mitochondrial ribosomal RNA (rrnS), and the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA region. Phylogenetic analyses were performed by using Maximum Parsimony (MP) analysis by PAUP. In addition, studies under SEM and LM were carried out on L3 larvae. All L3 individuals from E. anchoita, adults from S. magellanicus, and P. atriceps clustered in the same clade, well supported in the MP tree inferred from the mtDNA cox2, and rrnS gene sequences analyses. Further, the sequence alignments of L3 larvae and adults of C. pelagicum here obtained at the ITS-1 and ITS-2 regions of the rDNA matched the sequences of C. pelagicum previously deposited by us in GenBank. Nematode recruitment (Ro) was equal to 33.07 (7.20-91.14) L3 larvae for C. pelagicum in each penguin's meal of anchovy. The MP tree topologies obtained from mtDNA cox2 and rrnS genes demonstrated that specimens of Contracaecum L3 larvae from E. anchoita and C. pelagicum from S. magellanicus as well as from P. atriceps constitute a unique clade, well-distinct and supported from all the others formed by the Contracaecum spp. sequenced so far for these genes. Molecular markers are considered to be an effective tool to elucidate larval transmission. The Contracaecum L3 larval recruitment value showed that many worms fail to establish in the bird digestive tract, probably because they are below a critical size. Further work is needed to elucidate other factors (e.g., physiological, immunological) that control nematode populations in the penguin digestive tract.

Rhabdias esculentarum n. sp. (Nematoda: Rhabdiasidae) from green frogs of the Rana esculenta species complex in Italy: molecular evidence, morphological description and genetic differentiation from its congeners in frogs and toads.

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.

Genetic and morphological evidences for the existence of a new species of Contracaecum (Nematoda: Anisakidae) parasite of Phalacrocorax brasilianus (Gmelin) from Chile and its genetic relationships with congeners from fish-eating birds.

Contracaecum australe n. sp. is described from the Neotropic cormorant Phalacrocorax brasilianus in Chile based on morphology and the sequence analyses of multiple loci, i.e., mitochondrial cytochrome oxidase 2, mtDNA cox-2, the small subunit of the mitochondrial ribosomal RNA gene, rrnS, and the ITS-1 and ITS-2 regions of nuclear ribosomal DNA. Moreover, sequence analysis of the same genes was carried out on the morphospecies Contracaecum chubutensis Garbin et al. (2008) from Phalacrocorax atriceps. Further, genetic relationships are presented between C. australe n. sp. and C. chubutensis with respect to the related congeners from fish-eating birds previously characterized genetically on the same genetic markers, i.e., Contracaecum rudolphii A, B, C, D, and E, Contracaecum septentrionale, Contracaecum microcephalum, Contracaecum bioccai, Contracaecum pelagicum, Contracaecum micropapillatum, Contracaecum gibsoni, and Contracaecum overstreeti. Several phylogenetic analyses (MP, NJ, and BI) inferred from mitochondrial genes (cox-2 , rrnS) were congruent in depicting C. australe n. sp. and C. chubutensis as forming distinct clades, highly supported, from the remainder of the Contracaecum taxa considered; thus, it validates their specific status. Further, analyses of the ITS-1 and ITS-2 sequence data of C. australe n. sp. and C. chubutensis supported their distinction with respect to the 2 sibling species, C. rudolphii D and C. rudolphii E, previously detected from Phalacrocoracidae of Australia. Morphological analysis and the differential diagnosis of male specimens of C. australe n. sp. enabled the detection of differences in a number of characters, including spicule length, peculiar shape of male tail, paracloacal papillae disposition, and shape and bifurcation depth of interlabia. According to the genetic and morphological results obtained, the erection of a new taxon from fish-eating birds of the Austral region is given and its formal description is presented. Phylogenetic trees support both C. australe n. sp. and C. chubutensis as being included in the same clade with the previously detected species from cormorants, i.e., C. rudolphii A, B, C, and C. septentrionale. The finding of C. australe n. sp. and C. chubutensis parasites of Ph. brasilianus and Ph. atriceps, respectively, appears to support a host-parasite association between the C. rudolphii A, B, and C, C. septentrionale, C. chubutensis, and C. australe n. sp. and different species of cormorants belonging to Phalacrocorax.