Molecular characterization of Maccallumtrema xiphiados (Trematoda: Azygiida) and Molicola sp. (Cestoda: Trypanorhyncha) infecting commercial frozen slices of Atlantic swordfish.

Muscle gross lesions, associated to parasites, were routinely found during self-inspection in a Spanish fish plant processing Atlantic swordfish. To determine the taxonomic status of these parasites, molecular analysis was performed based on 18S, ITS1, 5.8S, ITS2 and 28S rDNA sequences, obtaining a consensus sequences of 4581 bp for the cestode and 4200 bp for the trematode. Taxonomic affiliation was determined by phylogenetic analysis of combined SSU + LSU rDNA regions using maximum likelihood models. Molecular characterization allows us to identify the trematode Maccallumtrema xiphiados and the cestode Molicola sp. infecting the musculature of the Atlantic swordfish. Both parasites are responsible of significant economic loss to fish industry due to commercial rejection of parasitized products.

A Minchinia mercenariae-like parasite infects cockles Cerastoderma edule in Galicia (NW Spain).

The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni- and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae-like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae-like parasites infecting molluscs outside the United States is confirmed.

Microsporidians xenomas of anglerfish from NE Atlantic waters.

The presence of emergent visible parasites at commercial valuable fish species is increasingly causing problems at fisheries and seafood industries. Microsporidians have been previously reported to appear forming apparent xenomas complexes in anglerfish species, but no effort has been carried out to simultaneously integrate epidemiological data, phenotypic, genotypic and fine structural characterizations in the same parasite sample. In this work, specimens of Lophius budegassa and Lophius piscatorius from NE Atlantic waters were sampled and examined to provide information about specific site of infection and demographic data of two groups of different sizes of xenomas present at both fish species. Histological descriptions and scanning and transmission electron microscopy were carried out on fresh spores of Lophius budegassa for ultrastructural studies. In both types of xenomas, it was observed simultaneously the microsporidian genus Spraguea in the form of two different types of spores. Molecular analyses of both xenomas from the two fish species, based on the small subunit ribosomal DNA gene, were also performed to genetically support the morphological diagnostic provided.

Hyperspora aquatica n.gn., n.sp. (Microsporidia), hyperparasitic in Marteilia cochillia (Paramyxida), is closely related to crustacean-infecting microspordian taxa.

The Paramyxida, closely related to haplosporidians, paradinids, and mikrocytids, is an obscure order of parasitic protists within the class Ascetosporea. All characterized ascetosporeans are parasites of invertebrate hosts, including molluscs, crustaceans and polychaetes. Representatives of the genus Marteilia are the best studied paramyxids, largely due to their impact on cultured oyster stocks, and their listing in international legislative frameworks. Although several examples of microsporidian hyperparasitism of paramyxids have been reported, phylogenetic data for these taxa are lacking. Recently, a microsporidian parasite was described infecting the paramyxid Marteilia cochillia, a serious pathogen of European cockles. In the current study, we investigated the phylogeny of the microsporidian hyperparasite infecting M. cochillia in cockles and, a further hyperparasite, Unikaryon legeri infecting the digenean Meiogymnophallus minutus, also in cockles. We show that rather than representing basally branching taxa in the increasingly replete Cryptomycota/Rozellomycota outgroup (containing taxa such as Mitosporidium and Paramicrosoridium), these hyperparasites instead group with other known microsporidian parasites infecting aquatic crustaceans. In doing so, we erect a new genus and species (Hyperspora aquatica n. gn., n.sp.) to contain the hyperparasite of M. cochillia and clarify the phylogenetic position of U. legeri. We propose that in both cases, hyperparasitism may provide a strategy for the vectoring of microsporidians between hosts of different trophic status (e.g. molluscs to crustaceans) within aquatic systems. In particular, we propose that the paramyxid hyperparasite H. aquatica may eventually be detected as a parasite of marine crustaceans. The potential route of transmission of the microsporidian between the paramyxid (in its host cockle) to crustaceans, and, the 'hitch-hiking' strategy employed by H. aquatica is discussed.

Nyctiphanes couchii as intermediate host for Rhadinorhynchus sp. (Acanthocephala, Echinorhynchidae) from NW Iberian Peninsula waters.

In the mesozooplanktonic community of the coastal upwelling system of the Ría de Vigo (NW Spain), the euphausiid Nyctiphanes couchii has been identified for the first time in temperate waters of the NE Atlantic as the intermediate host for cystacanths of Rhadinorhynchus sp. Parasites were identified using morphological characters described in 20 cystacanths. The hooks of the proboscis were arranged in 14 rows of 26 hooks each, while the hooks of the basal circle were only slightly erected and were longer than remaining spines. A maximum-likelihood estimation (ML) tree inferred from the 18S rRNA data set of Palaeacantocephala revealed that our specimens belong to a highly supported clade with Rhadinorhynchus sp., Pararhadinorhynchus sp. and Transvena annulospinosa. Nonetheless, our morphological and phylogenetic analyses suggested that the status of Rhadinorhynchus pristis should be re-examined. The prevalences of parasites were 0.0019% and 0.0001% for frontal and coastal summer communities, and 0.0068% and 0.0008% for coastal and oceanic autumn communities, respectively. The presence of these cystacanths in different mesozooplankton communities throughout the study suggests that the recruitment of parasites may be affected by the oceanography.

The occurrence of haplosporidian parasites, Haplosporidium nelsoni and Haplosporidium sp., in oysters in Ireland.

The phylum Haplosporidia is a group of obligate protozoan parasites that infect a number of freshwater and marine invertebrates. Haplosporidian parasites have caused significant mortalities in commercially important shellfish species worldwide. In this study, haplosporidia were detected in Pacific oysters Crassostrea gigas originating in Ireland and were subsequently identified independently in laboratories both in Ireland and in Spain as Haplosporidium nelsoni. In Ireland, H. nelsoni plasmodia were also observed in the heart tissue of a single Ostrea edulis. A range of techniques including heart smear screening, histology, standard polymerase chain reaction (PCR), direct sequencing and in situ hybridisation with an H. nelsoni specific DNA probe were carried out to confirm diagnosis. This is the first reporting of H. nelsoni in oysters in Ireland and this is the first reporting of the detection of this haplosporidian in O. edulis. In Ireland, another haplosporidian was also observed in a single O. edulis during heart smear screening. PCR and DNA sequencing were carried out and indicated the presence of a Haplosporidium sp., most likely Haplosporidium armoricanum. The low prevalence and intensity of infection of both haplosporidian species in Irish C. gigas and in particular O. edulis may indicate that their presence is inconsequential.

Nyctiphanes couchii as intermediate host for the acanthocephalan Bolbosoma balaenae in temperate waters of the NE Atlantic.

Cystacanths of the acanthocephalan Bolbosoma balaenae (Gmelin, 1790) were found encapsulated in the cephalothorax of the euphausiid Nyctiphanes couchii (Bell, 1853) from temperate waters in the NE Atlantic Ocean. Euphausiids were caught in locations outside the Ría de Vigo in Galicia, NW Spain, and prevalence of infection was up to 0.1%. The parasite was identified by morphological characters. Cystacanths were 8.09 ± 2.25 mm total length (mean ± SD) and had proboscises that consisted of 22 to 24 longitudinal rows of hooks, each of which had 8 or 9 hooks per row including 2 or 3 rootless ones in the proboscis base and 1 field of small hooks in the prebulbar part. Phylogenetic analyses of 18S rDNA and cytocrome c oxidase subunit I revealed a close relationship with other taxa of the family Polymorphidae (Meyer, 1931). The results extend northwards ot the known distribution of B. balaenae. Taxonomic affiliation of parasites and trophic ecology in the sampling area suggest that N. couchii is the intermediate host for B. balenae, and we suggest that the whales Balaenoptera physalus (Linnaeus, 1758) and B. acutorostrata (Lacepède, 1804) are its definitive hosts. This life cycle is probably completed with or without paratenic hosts.

Observations raise the question if the Pacific oyster, Crassostrea gigas, can act as either a carrier or a reservoir for Bonamia ostreae or Bonamia exitiosa.

This study investigated the ability of the Pacific oyster, Crassostrea gigas, to act as a carrier or reservoir of the protistan Bonamia ostreae. Studies were carried out independently in Ireland and in Spain. Naïve C. gigas were exposed to B. ostreae both in the field and in the laboratory via natural exposure or experimental injection. Naïve flat oysters, Ostrea edulis, were placed in tanks with previously exposed C. gigas. Oysters were screened for B. ostreae by examination of ventricular heart smears and by polymerase chain reaction (PCR) screening of tissue samples (gill and/or heart) and shell cavity fluid. PCR-positive oysters were further screened using histology and in situ hybridization (ISH). B. ostreae DNA was detected in the tissues and/or shell cavity fluid of a small number of C. gigas in the field and in the laboratory. B. ostreae-like cells were visualized in the haemocytes of 1 C. gigas and B. ostreae-like cells were observed extracellularly in the connective tissues of 1 other C. gigas. When C. gigas naturally exposed to B. ostreae were held with naïve O. edulis, B. ostreae DNA was detected in O. edulis; however, B. ostreae cells were not visualized. In Spain, B. exitiosa DNA was also detected in Pacific oyster tissues. The results of this study have important implications for C. gigas transfers from B. ostreae-endemic areas to uninfected areas and highlight B. ostreae and B. exitiosa's ability to survive extracellularly and in other non-typical hosts.

Myxosporean infection in frozen blocks of Patagonian hakes.

Numerous pseudocysts of the myxosporean genus Kudoa were found infecting the body musculature of commercial frozen blocks of Patagonian hakes of the species Macruronus magellanicus, Merluccius australis, and Merluccius hubbsi. Pseudocysts consisted of numerous diminutive quadrate spores with four polar capsules. The small subunit ribosomal DNA was amplified, cloned, and sequenced for phylogenetic study. Minimum evolution and maximum parsimony analyses placed the resulting sequences in a clade with Kudoa species of Patagonian hakes, K. rosenbuschi and K. alliaria, with bootstrap values of 99%. Molecular and morphological results indicated that the above species were the same, with some differences based on host-related factors. Minor muscular damage appeared as a focal distortion of host fiber architecture to accommodate the parasite. The mild host response consisted of different stages of encapsulation. In cases of concurrent heavy infections, these fish are unappealing as food, due to the parasite's potential effect on the market's perception of macroscopic, unsightly cysts in fish musculature.

Occurrence of recombinant genotypes of Anisakis simplex s.s. and Anisakis pegreffii (Nematoda: Anisakidae) in an area of sympatry.

The anisakid nematode populations collected from fish and stranded cetaceans along from Iberian Peninsula waters were morphologically identified as corresponding to the Anisakis simplex complex. In order to realise their molecular identification and to analyse the extent of genetic variation, the entire ITS (ITS1, 5.8S rDNA gene and ITS2) and the mitochondrial small subunit of rRNA were pcr-amplified and sequenced. Digestions of the amplified its region with HinfI and HhaI allowed the identification of three different genotypes, belonging to A. simplex s.s., A. pegreffii and a yet not described recombinant genotype. The ITS sequences of the recombinant genotypes showed the presence of heterozygotes C/T at position 240 and 256 of the aligned sequence. Otherwise, the analysis of mtDNA sequences showed the existence of a different parental origin for recombinant genotypes. In order to check if they can be the products of a polymorphism normally occurring both in A. pegreffii and in A. simplex s.s., and/or the existence of an incomplete concerted evolution, three samples were also collected as controls in isolated geographic areas, where sympatric coexistence between A. simplex s.s. and A. pegreffii does not occur. The results supports the hypothesis that the recombinant individuals may be a product of interspecific hybridisation, and describe the Iberian Peninsula waters as a hybrid zone for the two sibling species.

Observations on associated histopathology with Aggregata octopiana infection (Protista: Apicomplexa) in Octopus vulgaris.

Gamogony and sporogony of Aggregata octopiana were commonly observed during histological examination of the digestive tract of wild Octopus vulgaris from Ria de Vigo (NW Spain). A. octopiana infected noncuticularized caecum and intestine, and cuticularized oesophagus and crop. Infection was also observed in the gills and in covering mesenterium, mainly of the digestive gland and gonad. Histological and ultrastructural lesions associated with A. octopiana included host cell hypertrophy with nuclear displacement, inflammation, phagocytosis, ulceration and destruction of organ architecture. The possible existence of a malabsorption syndrome in the host is deduced.

Anisakid infection in the European shag Phalacrocorax aristotelis aristotelis.

A total of 100 specimens of the European shag Phalacrocorax aristotelis aristotelis were examined for anisakid infections in the south-east North Atlantic. The taxonomic status of the anisakid nematodes was studied by structural analysis using light and scanning electron microscopy. Two species, Contracaecum septentrionale and C. rudolphii A (Nematoda: Ascaridoidea), occurred in the European shag with high values of prevalence and mean intensities of infection. These constitute new host records for both parasites, widening their known geographical distribution. Although the histopathology associated with the infections indicated that the anisakids did not have the potential to cause bird death, the parasites are capable of contributing to and hastening avian mortality in the case of heavily-parasitized cormorants and when stressed by xenobiotics and/or through a synergetic effect by other bioagressors.

Element concentration variability in the whaleworm Anisakis simplex s.l.

Concentrations of the elements C, N, Fe, Na, K, Ca and Mg were analysed by chromatography and atomic absorption spectrometry in the whaleworm Anisakis simplex sensu lato. Nearly all mean inorganic element concentrations measured were higher in adult worms than in larval forms. Similarly, adults contained significantly higher nitrogen (i.e. lower C/N ratio values) than either eggs or larvae. Evidence for the significance of the parasitic life cycle stage and the systematic position of their hosts on the element concentration variability is discussed.

Fitness of the marine parasitic nematode Anisakis simplex s. str. in temperate waters of the NE Atlantic.

In temperate waters of the NE Atlantic, third-stage larvae of Anisakis simplex sensu stricto collected from 3 paratenic host species were identified by restriction fragment length polymorphisms. The condition of wild larval infrapopulations was assessed by examining morphometric and growth characteristics. The differentiation patterns and the excretory/secretory products of larvae grown in vitro were also examined. An extensive morphometric, growth and differentiation variability was found between parasite larvae collected from different paratenic host sources. Nematode infrapopulation larvae from the squid comprise those smaller individuals with the lowest values of survival rates and moult success. It may be then concluded that the fitness of A. simplex s. str. larvae is not the best possible in the squid, which impaired the competitiveness of the parasite and its chances of developing into an adult. This suggests that the microenvironments impaired by the paratenic host may provide larval infrapopulations with unique ecological factors probably influencing its recruitment to the final host populations.

Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective.

A total of 2,673 fresh specimens of cephalopod and fish representing 35 species were obtained from commercial local fisheries in Galician waters (NW Spain). They were examined for anisakid nematodes by digestion of the muscle and elution of the viscera and whole body cavity. All larval nematodes recovered were identified by light microscopy and multilocus electrophoresis as belonging to the species Anisakis simplex sensu stricto and A. pegreffii. Encysted larvae mostly occurred in the viscera but were also found in the flesh of squid and fish. Demographic values for larval nematodes are discussed in relation to host preferences and the ecological niche of both anisakid species at the sampling area. Primary recommendations are also expressed concerning the effects of current fishing and aquaculture practices on the Anisakis problem.

Genetic divergence and reproductive isolation between Anisakis brevispiculata and Anisakis physeteris (Nematoda: Anisakidae)s.

In order to assess the taxonomic status of Anisakis brevispiculata Dollfus, 1966 population samples of this taxon from central and south-eastern Atlantic ocean were compared at 22 enzymatic loci with samples belonging to Anisakis physeteris Baylis, 1923 from the Mediterranean sea and central-eastern Atlantic ocean. Very low interpopulational genetic divergence was observed both within A. brevispiculata (average D(Nei) = 0.008) and within A. physeteris (D(Nei) = 0.009) despite the geographic distance among the samples, indicating high levels of gene flow in both taxa. On the other hand, the average genetic distance between A. brevispiculata and A. physeteris was found to be D(Nei) = 0.80, a value generally observed between well differentiated congeneric species. The reproductive isolation between A. brevispiculata and A. physeteris is indicated by the following observations: (1) no F(1) hybrids or recombinant genotypes were until now observed; and (2) the two Anisakis species do not seem to share their definitive hosts. The main definitive host of A. brevispiculata is the pygmy sperm whale (Kogia breviceps), while for A. physeteris it is the sperm whale (Physeter catodon). Only adult males differ slightly in spicule length, while females and larval stages are not differentiated morphologically. Both A. brevispiculata and A.physeteris show a type II larva. The correct recognition of A. brevispiculata from A. physeteris and from other Anisakis species studied, in either sexes and at any life stage, is made easy by allozyme markers (e.g. Icdh, Gapdh, Sod-1, Np, Aat-2, Adk-2, fEst-2, PepB, PepC-2, Mpi). Diagnostic keys, which can be used for routine identification in the field of these Anisakis worms, based on genetic markers, are given.

Aggregata sagittata n. sp. (Apicomplexa: Aggregatidae), a coccidian parasite from the European flying squid Todarodes sagittatus (Mollusca: Cephalopoda).

A new species of the coccidian genus Aggregata Frenzel, 1885 (Apicomplexa, Aggregatidae) is described from the digestive tract of Todarodes sagittatus (Lamarck), an ommastrephid squid that lives in temperate waters in the northeastern Atlantic Ocean. Using light microscopy. oöcysts were 225-333 microm in length and 220-323 microm in width. Each oöcyst contained 600-1,000 sporocysts. Sporocysts were 16-18 microm in length and 15-17 microm in width, with a smooth surface and thick wall. Each sporocyst contained 4-8 sporozoites each with a length of 11-13 microm. This paper presents the description of a new Agregata species in a nerito-oceanic flying squid. Moreover. it extends the distribution of Aggregata to oceanic cephalopod host species. A. sagittata represents the third named species in the genus Aggregata in European waters and the seventh worldwide.

Elemental analysis of cetacean skull lesions associated with nematode infections.

The elemental composition of both healthy and eroded cetacean skulls associated with nematode infections was evaluated. A total of 27 samples of eroded and non-eroded prepared museum cetacean skulls were characterised by elemental (CHN), X-ray fluorescence, and X-ray diffraction methods. The inorganic composition and crystal line structure (hydroxylapatite-like minerals) were similar for both types of skull samples, but the CHN values clearly differed. The results suggest that the carbon-rich fraction is lost in eroded areas, probably as a result of glycosaminoglycan-degrading Crassicauda enzymes.

Larval nematodes (Spiruroidea: Cystidicolidae) in Octopus vulgaris (Mollusca: Cephalopoda: Octopodidae) from the northeastern Atlantic Ocean.

Larval nematode parasites (Spiruroidea: Cystidicolidae) are recorded for the first time in Octopus vulgaris Cuvier, 1797 in the northeastern Atlantic Ocean. Prevalence was 16% and mean intensity was 1.46 worms/host. Body length of larval nematodes ranges from 8.3 to 9.3 mm, with a distance from the anterior end to nerve ring from 187.5 to 200 microm, and to excretory pore 194.6-350 microm. Anatomical characteristics, such as deirid, nerve ring, cephalic alae, excretory pore, pseudolabia amphids, sclerotized protuberance, and anus, examined using light microscopy (LM) or scanning electron microscopy (SEM), are illustrated. The nematode was designed as a cystidicolid "Type A" larva. The hemocytic infiltration present in the host tissue around the nematode capsule and the mechanical compression in the infected organs denote the pathogenicity of this nematode. In the study area, O. vulgaris may play the role of an intermediate or paratenic host in the nematode life cycle.