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8 pages, 6 figures, 1 table, During the last years the presence of parasites in commercial fish species has increased becoming an emergent major sanitary problem. For seafood companies the control of this biological hazard is turning into a priority issue, as quality of the products is now being seriously compromised. The swordfish Xiphias gladius, as one of the most important commercial species among European markets, has been inspected for the presence of pennellids. A total of 214 mesoparasitic copepods were sampled for epidemiology and genetic analysis, 5 pennellid cephalothoraxes’ for morphological and genetic studies and 50 slices of parasitized swordfishes were examined for internal lesions and diagnostics on the basis of morphological characters. All hosts were captured in Atlantic waters. Pennella instructa was morphologically identified as the only metazoan species in the 5 cephalothoraxes. Morphological characters and internal and external lesions caused by P. instructa were acutely described. Prevalence and mean Intensity of infection were determined for the total fishes parasitized. Copepods were molecularly characterized and the phylogenetic relationships were explored based on 18S and 28S rDNA sequences. Phylogenetic analyses using maximum likelihood models were performed including pennellid external portions and cephalothoraxes (morphologically identified as P. instructa). All pennellid samples were clustered into the same clade with bootstrap values of 100% for both genes sequenced. The prevention of rejections due to these parasites must be the first step to ensure safer and high quality standard products to final consumers. Monitoring actions and proactive self-inspections including preventive and corrective measures should be more intensively integrated into HACCP systems of seafood companies, Xunta de Galicia has funded this work under Projects INCITE‐07MMA015CT, IN841C, 10TAL001CT and 10TAL033E. M. Llarena‐Reino thanks Fundação para a Ciência e a Tecnologia for its financial support under Grant SFRH/BD/45398/2008

© The Author(s)., [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., This work has been partially supported by the Istituto Zooprofilattico Sperimentale della Sicilia – granted project "Indagine epidemiologica sulla prevalenza di allergie da Anisakis nella Regione Sicilia”.

To screen the presence of Anisakidae third stage larvae (L3) in fish, fast methods such as the visual inspection and candling have been widely used by the industry over the last 50 years, and they are regulated by the European Parliament and the Council. These methods are ineffective to detect L3 embedded in fish muscles, consequently alternative methods, such as the artificial digestion (AD) and the UV-Press (UVP) are increasingly applied, but their performance needs to be evaluated. The aims of the present work were: 1) to compare the performance of AD and UVP. methods by a Ring Trial (RT) involving highly experienced laboratories; and 2) to evaluate the potential transferability of the best performing method to the industry by a collaborative study involving industrial partners (β-testing). For RT, each participating laboratory (n = 5) received 6 samples of 100 g of fish fillets spiked with 1 L3 (1 sample), 3 L3 (2 samples), 7 L3 (2 samples), and a negative control sample (without L3). In each positive sample, there were live Anisakis pegreffii L3 collected from a naturally infected fish. The result evaluation was based on the agreement between the number of reported and the number of spiked L3. No false positive sample was detected. The L3 number detected by the UVP method showed higher (90%) level of agreement with the number of spiked L3 than the number of L3 detected by the AD method (83.3%); however, no significant difference in terms of accuracy (p = 0.32) was detected when the two methods were compared. Moreover, considering only the presence/absence of L3 in the samples, the UVP reached 100% of accuracy and 100% of sensitivity; whereas, AD showed 98% of accuracy and 96% of sensitivity. The variability of the UVP method was lower than that of the AD method, indicating a better reproducibility. On the basis of the RT results, the UVP method was selected for the β-testing. Each industrial partner (n = 3) received 15 samples of 100 g of fish fillet spiked with 1 L3 (2 samples), 2 L3 (2 samples), 3 L3 (2 samples), 4 L3 (2 samples), 5 L3 (2 samples) and 6 L3 (2 samples) and three negative control samples (without L3). The number of L3 counted in 34 out of 45 samples (75.6%) by the UVP method was in agreement with the number of spiked L3. One company reached 93.3% of agreement; whereas the other two companies reached an agreement of 66.7%. Two false negative results were found; whereas, no false positive results were obtained. Moreover, at the industrial level, considering only the presence/absence of larvae in the samples, the UVP reached 97% of accuracy, 94.4% of sensitivity, and 100% of specificity. However, the UVP method, in spite of its accuracy, needs further investigations to provide new time −temperature combinations that could allow a reduction of the testing time and its integration in the fishing deck.

10 pages, 2 tables, 2 figures, In recent years, scientific evidence from clinical case studies has been accumulated to suggest that consumption of homemade raw anchovies (Engraulis encrasicolus) in vinegar (referred to locally as “boquerones en vinagre”) represented the main potential risk source for anisakiasis and IgE sensitization to Anisakis in Spain. Conversely, most epidemiological surveys for this fish species in fishing grounds along the Spanish coast and in Spanish markets showed parasite-free fish lots or parasite prevalence below 10%. In order to update the anchovy infection data and to re-evaluate the importance of this species as a potential source of risk exposure to zoonotic and allergy diseases, 956 anchovies collected by Spanish commercial fishing vessels operating in NE Atlantic fishing grounds were analyzed, using a high performance methodology. Results revealed an overall mean prevalence for zoonotic nematodes (genera Anisakis and Hysterothylacium combined) of 64.33% (14.75% in the flesh). Infection values reached 87% in one of the lots. Prevalence in anchovy muscle was 28.65%, which represents the highest value of parasitization of the flesh ever reported for European stocks of this species. An enhanced risk communication plan focused on control measures, such as removal of anchovy viscera by retailers, and educational campaigns stressing the importance of freezing anchovies that are intended for raw consumption, may clearly contribute to avoid this emergent biohazard in seafood, The EU-granted PARASITE Project (GA312068) supported this work

12 pages, 9 figures, 2 tables, 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., Xunta de Galicia has funded this work under Projects INCITE-07MMA015CT, IN841C, 10TAL 001CT and 10TAL033E. M. Llarena-Reino thanks Fundação para a Ciência e a Tecnologia for its financial support (Grant SFRH/BD/45398/2008).

11 pages, 6 figures.-- This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License, Cephalopods and their metazoan parasites have coevolved in wild fisheries for many years. In fact, helminth larvae and parasitic copepods have been recorded in cephalopods worldwide. This is not surprising considering the important role cephalopods play in the transfer of energy and contaminants in marine food webs. Nerito-oceanic ommastrephid squids are by far the most noticeable trophic bridge for helminth parasites in the marine realm, coastal octopus, and cuttlefish serving as primary host for crustaceans. Although it is highly likely that parasitic infections occurred, relatively little is known about the pathogenic potential of metazoan parasites in naturally infected cephalopods. It is stated that heavy parasitic infections may probably cause host morbidity or poor condition but signs of disease are singularly rare with very few specimens exhibiting disease conditions. Unfortunately, neither robust scientific evidence nor available material is available to support this statement. It is more likely that metazoans may deplete energy stores of infected cephalopods, which are directed toward tissue repair and the host’s defense mechanisms. Parasitic infection may thus be considered an environmental stressor and as such a source of uncertainty in the evaluation of the potential productivity of cephalopod populations

6 pages, 2 tables, 2 figures, We carried out a comparison of three different tools for determining the risk of Anisakis exposure in fish: i) a risk ranking tool registered as a trade mark (FPR standard) based on a previous risk categorization scheme (Site, abundance, density and exposure, SADE), ii) the current visual method for fish inspection (V) and iii) the UV-press/peptic digestion (UV-P/PD) method. We used 2377 fish from 9 species and three important ICES fishing areas. The percentage of rejection for each fish lot was calculated according to the criteria established for the three different methods as follows: V, presence of visible parasites in the viscera and/or abdominal cavity; UV-P/PD, presence of parasites in the fish flesh; FPR scoring for parasite risk, individuals obtaining the classification of “poor”. The highest rejection rate was for UV-P/PD method, rejecting all the fish that harbor just one Anisakis in the edible part of the fish. Follow the rejection rate of fish under visual inspection, being the lowest that catalogued using the FPR standard. Visual inspection penalizes species with far higher rates of visceral than muscular parasitation, such as the blackbellied angler. Conversely, fish individuals preliminarily accepted by visual inspection would be rejected by FPR, thus minimizing the risks for consumers in species, such as in the case of the European hake, with high flesh infection. In some cases, the edible part exceeded the number of parasites found in the abdominal cavity and visceral organs. Overall, the results obtained after combining various geographic areas and fish species clearly suggest that a risk ranking tool represents a better trade-off between a best-value for money approach and the best-assurance for fish quality and safety approach than current inspection methods FPR also allows the possibility of an objective, transparent and efficient classification of the fish lots in 5 different categories from “poor” to “excellent”. As a conclusion, the FPR scheme should be routinely used within HACCP programs to facilitate the risk standardization and communication to improve the fish quality and guarantee healthier products, The EU-granted PARASITE Project (GA312068) supported this work

10 pages, 1 figure, 3 tables, This study investigates the occurrence of anisakids and raphidascarids in commercial fish from Balearic Sea (Western Mediterranean). A total of 335 fish including 19 black anglerfish (Lophius budegassa), 33 white anglerfish (L. piscatorius), 129 European hake (Merluccius merluccius), 30 red mullet (Mullus barbatus), and 124 striped mullet (M. surmuletus) were examined using enzymatic digestion. A total of 948 nematode larvae were isolated (prevalence 52.53%) being the highest prevalence observed in striped mullet. Forty-six larvae were identified using molecular analyses which included PCR and sequencing of the 629-bp fragment of mitochondrial cox2 gene region. Anisakis pegreffii (80.43%), A. physeteris (8.69%), Hysterothylacium fabri (6.52%), and A. simplex (4.35%) were detected based on molecular analyses of larvae. Total nematode prevalence was positively correlated with weight, length, condition factor, and maturity stage of the host and also with fishing ground depth. Statistical differences between total nematode prevalence and geographical sector of capture were observed when fishing hauls were grouped according to the abundance of sperm whales or common bottlenose dolphins. The results also corroborate that fishing water depth may play an important role in anisakid and raphidascarid parasitization.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l’Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l’Ebre.

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.

MicroRNAs (miRNAs) are small (∼22nt) non-coding regulatory single strand RNA molecules that reduce stability and/or translation of sequence-complementary target. miRNAs are a key component of gene regulatory networks and have been involved in a wide variety of biological processes, such as signal transduction, cell proliferation and apoptosis. Many miRNAs are broadly conserved among the animal lineages and even between invertebrates and vertebrates. The European flat oyster Ostrea edulis is highly susceptible to infection with Bonamia ostreae, an intracellular parasite able to survive and proliferate within oyster haemocytes. Mollusc haemocytes play a key role in the immune response of molluscs as main cellular effectors. The roles of miRNAs in the immune response of O. edulis to bonamiosis were analysed using a commercial microarray platform (miRCURY LNA™ v2, Exiqon) for miRNAs. Expression of miRNAs in haemocytes from oysters with different bonamiosis intensity was compared. Differential expression was detected in 63 and 76 miRNAs when comparing heavily-affected with non-affected oysters and with lightly-affected ones, respectively. Among them, 19 miRNAs are known to be linked to immune response, being responsible of proliferation and activation of macrophages, inflammation, apoptosis and/or oxidative damage, which is consistent with the modulation of their expression in oyster haemocytes due to bonamiosis.

Bonamiosis constrains the flat oyster industry worldwide. The protistan species Bonamia ostreae had been considered solely responsible for this disease in Europe, but the report of B. exitiosa infecting Ostrea edulis 5 yr ago in Galicia (NW Spain), and subsequently in other European countries, raised the question of the relevance of each species in bonamiosis. The spatial distribution of B. exitiosa and B. ostreae in Galicia was addressed by sampling 7 natural O. edulis beds and 3 culture raft areas, up to 3 times in the period 2009 to 2010. B. ostreae infected flat oysters in every natural bed and every raft culture area. True B. exitiosa infections (histological diagnosis) were detected in every raft culture area but only in 2 natural beds, i.e. in 4 rías. PCR-positive results for B. exitiosa were recorded in 4 out of 5 beds where true infections were not found, thus the occurrence of B. exitiosa in those 4 beds cannot be ruled out. Additionally, 4 cohorts of hatchery-produced oyster spat were transferred to a raft to analyse Bonamia spp. infection dynamics through oyster on-growing. The highest percentages of oysters PCR-positive for both Bonamia spp. were recorded in the first months of on-growing; other peaks of PCR-positive diagnosis were successively lower. Differences in the percentage of PCR-positive cases and in the prevalence of true infection between B. exitiosa and B. ostreae through on-growing were not significant. Our results support that B. exitiosa is adapted to infect O. edulis in the Galician marine ecosystem.

SUMMARYThe 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.

7 pages, 2 figures, 3 tables, This study aims to investigate the occurrence of nematode larvae in commercial cephalopods in the Western Mediterranean Sea. A total of 202 animals comprising 123 broadtail shortfin squid (Illex coindetii), 34 European squid (Loligo vulgaris) and 45 common octopus (Octopus vulgaris) were examined using enzymatic digestion. A total of 31 larvae were isolated (prevalence: 14.6%) and identified using molecular analyses which included PCR and sequencing of the ITS (ITS1-5.8S rDNA-ITS2) region. Phylogenetic tree inferred from ITS sequences yielded supported relationships for Anisakis (P: 12.2%) and Hysterothylacium species (P: 4.1%). All parasites were found parasitizing I. coindetii and, as expected, A. pegreffii presented the highest prevalence (11.4%). A. physeteris was also found with a lower prevalence (1.6%) but confirming the role of the broadtail shortfin squid as paratenic host and, its potential host for anisakidosis transmission. A hybrid larva between Anisakis simplex and A. pegreffi was also identified. All Anisakis larvae were found within the visceral cavity; in contrast most of the Hysterothylacium larvae were isolated from the mantle. A significant correlation was found between total nematode prevalence and depth, explained by the presence of larger broadtail shortfin squids inhabiting deeper depths. Therefore, the results obtained in the present study improve the knowledge of the occurrence of Anisakis and Hysterothylacium species in the I. coindetii from the Spanish Mediterranean Sea highlighting the importance of considering I. coindetii as a potential hazard for humans if it is consumed raw or not well cooked and the need of further research in other cephalopods.

7 pages, 1 figure, The use of marine parasites as biological tags in fish stock studies over the last 60 years became a widely accepted method commissioned by scientific and fisheries agencies, and the results were included in species reports of fishery management plans. Much effort has been made to understand the ecological role and applicability of a wide diversity of parasite fauna as tags across major marine areas and oceans, especially for the main fish stocks and fishing grounds. Herein, we explore biobanking and genetic marker challenges as tools to be used for fish stock discrimination. First of all, we discussed about well-recognized major gaps identified by researchers, managers and end-users that improve the usefulness of parasites as tags. Secondly, we suggest the potential of under-exploited biobanking and molecular markers as promising technical solutions, which could play a future leading role ensuring such a priori high discriminatory power of marine fish populations based on parasite data.

The European flat Ostrea edulis is highly susceptible to infection by the protozoan Bonamia ostreae and Bonamia exitiosa, intracellular parasites able to survive and proliferate within the oyster haemocytes. The parasite, once phagocytosed by the haemocyte, the main cellular effector of the immune system, appears to have some counter mechanism that turns off the haemocyte's metabolic destructive capacity, so that the parasite survives within the cell. To further understand the molecular basis of the immune response of the flat oyster against the bonamiosis, suppression subtractive hybridization and Q-PCR approaches were combined to identify genes involved in the development of the infection both in early and advanced phases. Four subtractive cDNA libraries were constructed and sequenced, obtaining a high number of ESTs that were seen to be up or down-regulated in the infection. A group of ESTs that play a role in the immune response, such as cytokines, stress proteins, eicosanoids, proteins implicated in phagocytosis and cell junction as well as in transcription signalling were identified and their expression was analysed at different infection levels by Q-PCR. The results here reported can help to enrich our understanding about the immune response of O. edulis against bonamiosis and improve our knowledge of the immune mechanisms of oysters.

SUMMARYThis 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.

The haplosporidian Bonamia exitiosa was found infecting the European flat oyster Ostrea edulis in the Galician coast (NW Spain), which represents the first report of this parasite along European waters. Histopathology and molecular characterization of the small subunit ribosomal DNA gene were performed to identify this species. Examination of histological sections showed two microcell types, the smaller one corresponding to Bonamia ostreae and the larger one to B. exitiosa. Phylogenetic analysis places the sequence herein reported in a clade with Bonamia species of the Southern hemisphere, namely B. exitiosa, B. roughleyi and Bonamia sp. from North Carolina and Chile. Subsequent PCR-RFLPs analysis showed a highly-endemiotopic infection by B. exitiosa, demonstrating the success of this haplosporidian to infect the European flat oyster in the Galician marine ecosystem even in concurrent infections with B. ostreae.

9 pages, 5 figures, 4 tables, A survey on perkinsosis was performed involving 15 locations scattered along the Galician coast (NW Spain) and four clam species with high market value (Ruditapes decussatus, Ruditapes philippinarum, Venerupis corrugata and Polititapes rhomboides). The prevalence of Perkinsus parasites was estimated by PCR using genus-specific primers. The highest percentage of PCR-positive cases for perkinsosis corresponded to clams R. decussatus and V. corrugata, while lower values were detected in R. philippinarum and no case was found in P. rhomboides. The discrimination of Perkinsus species was performed by PCR–RFLP and by a new PCR–DGGE method developed in this study. Perkinsus olseni was identified in every clam species, except in P. rhomboides, using both PCR–DGGE and PCR–RFLP. Additionally, Perkinsus chesapeaki was only detected by PCR–DGGE infecting two Manila clams R. philippinarum from the same location, reporting the first case in Galicia. P. chesapeaki identification was further confirmed by in situ hybridisation assay and phylogenetic analysis of ITS region and LSU rDNA., This study was funded by the Ministerio de Economía y Competitividad of the Government of Spain, through the project AGL2011-30449-C02-01.

19 páginas, 3 tablas, 3 figuras, The euphausiid Nyctiphanes couchii and an unidentified mysid have been found, for the first time, with third-stage larvae (L 3 ) of the Anisakis simplex complex in the mesozooplanktonic community of the coastal upwelling system in Galicia (NW Spain). Parasite larvae were molecularly identified using the internal tran- scribed spacer (ITS) region. The prevalence of these parasites in the euphausiid population was 0.0019%. The existence of parasites in a variety of mesozooplank- ton organisms suggests that the transmission routes of A. simplex sensu stricto and A. pegrefii are wider than expected. The results suggest that these two Anisakis species are not specific to their intermediate hosts. Finally, the recruitment of A. simplex complex may be affected by oceanography, differing under upwelling or downwelling conditions., This research was supported by ‘ Canaries – Iberian Marine Ecosystem Exchanges ’ [grant number CTM-2007-66408-CO2-01] and ‘ Larval Ecology ’ [grant number CTM-2011-25929]. The first author and A.R. were funded by Junta para la Ampliación de Estudios pre-Doctorales (Spanish National Research Council) co-financed with Fondo Social Europeo (ESF) funds

Perkinsosis is an infection of marine molluscs caused by the protistan parasites of the genus Perkinsus , which has been classified by the OIE as a disease that warrants notification. In the present study, we have applied a molecular genetic approach to develop an optional method for the specific identification of Perkinsus species. A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the rRNA ITS region was developed to identify and distinguish among Perkinsus species. A taxonomic key was established that allows successful identification of Perkinsus species using a single restriction enzyme ( Rsa I) to discriminate P. chesapeaki and P. marinus or by a combination of two endonucleases ( Rsa I plus Hinf I) to discriminate P. olseni and P. mediterraneus . In order to validate the RFLP assay, the PCR products were cloned and sequenced, and its phylogenetic affinity was determined. Phylogenetic analysis confirmed the specific identification carried out by RFLPs. Herein is the first report of P. olseni in Manila clams from the NW Adriatic Sea (Italy), which we identified by employing this method. The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species, and may aid in ongoing epizooetiological studies and diseases control programmes.

SUMMARY: Scanning Electron Microscopy (SEM) was used to study different topo-morphological characteristics of the architecture (cuticle, excretory pore, lips and adjacent structures, number and distribution patterns of caudal papillae and papillae-like structures) of the anisakid nematodes Anisakis brevispiculata and Pseudoterranova ceticola, parasites in the stomach of the pygmy sperm whale Kogia breviceps. SEM micrographs herein help to accurately define the above surface topographical features by adding a more adequate 3-D picture to the original descriptions of both parasitic species. In A. brevispiculata the entire body cuticle structure, well-differentiated paracloacal papillae and the wrinkle cuticle of the papillae are clear examples that enhance the above differentiation of structures as seen by LM or SEM. Similarly, in P. ceticola the cuticle striations, bulky cloacal lips, rectangular distribution pattern of distal papillae and the absence of a groove separating paracloacal papillae which are obliquely arranged are all different to those features previously described.

Bonamiosis is a disease affecting various oyster species and causing oyster mass mortalities worldwide. The protozoans Bonamia exitiosa and B. ostreae (Haplosporidia) are included in the list of notifiable diseases of the World Organisation for Animal Health as the causative agents of this disease. Although the geographic range of both species was considered different for years, both species are now known to co-occur in some European areas affecting the same host, Ostrea edulis, which strengthens the need of species-specific methods to unequivocally identify the species of Bonamia. An oligonucleotide probe for specific detection of B. exitiosa (BEX_ITS) was designed to be used in in situ hybridisation (ISH) assays. ISH assay with BEX_ITS probe showed species-specificity and more sensitivity than traditional histology to visualise the parasite inside host tissue. ISH assay showed that the oyster gonad was the area where the parasite was most frequently located, and was the exclusive organ of infection in some oysters. A recommendation arising from the study is that more than 1 organ (including gonad and gills) should be used for PCR-based diagnosis of B. exitiosa, to maximise the sensitivity.

The name 'microcells' is frequently used to refer to small-sized unicellular stages of molluscan parasites of the genera Bonamia (Rhizaria, Haplosporidia) and Mikrocytos (Rhizaria). Histological examination of Manila clams Ruditapes philippinarum revealed microcells in the connective tissue of adductor muscle, foot, mantle, gills, siphon and visceral mass. The clams had been collected from 4 beds on the coast of Galicia, Spain. The prevalence of these microcells ranged from 73 to 93% in surface clams and from 3 to 33% in buried clams. However, the detection of brown ring disease signs in clams from every bed prevented us from making the assumption that the microcells alone were responsible for clam mortality. PCR assays using primer pairs designed to detect Bonamia spp. and haplosporidians gave negative results, whereas positive results were obtained with primers for the genus Mikrocytos. A consensus sequence of 1670 bp of the ribosomal gene complex of the microcells was obtained. It contained a section of the 18S region, the whole first internal transcribed spacer, the 5.8S region, the second internal transcribed spacer and a section of the 28S region. Comparison of this sequence with those of M. mackini infecting Crassostrea gigas and Mikrocytos sp. infecting Ostrea edulis showed that the microcells of Galician clams were the most divergent among the compared parasites. This is the first report of a Mikrocytos-like parasite infecting Manila clams. Care must be taken to avoid the spread of this parasite through Manila clam transfers.

The highest shellfishery catch in Galicia (NW Spain) has traditionally been cockle Cerastoderma edule. The shellfish bed located in Lombos do Ulla (Ria de Arousa) used to be among those with the highest cockle production; however, cockle mortality rate increased sharply in this bed in April 2012, reaching 100% in May 2012. Salinity and temperature were discounted as potential causes of the mortality. Marteiliosis, which was first detected in February 2012 and reached 100% prevalence in April 2012, was identified as the most probable cause. Marteiliosis had never been detected in Galician cockles, but extensive surveillance of the Galician coast in May to July 2012 detected marteiliosis in most cockle beds of the Ria de Arousa, whereas it was not found in other rias; 2 mo later, the cockle catch in the Ria de Arousa became negligible. Examination of the aetiological agent of marteiliosis with light and transmission electron microscopy supported its assignation to the genus Marteilia; morphological features showed similarity, but not complete identity, with the recently described species M. cochillia Carrasco et al., 2013. Regarding its molecular characterisation, a consensus sequence of 4433 bp containing a partial sequence of the intergenic spacer region, the complete 18S rRNA gene and a partial sequence of the first internal transcribed spacer region was obtained. The obtained sequences were compared with those available for Marteilia spp. and other Paramyxida. Molecular data support that this parasite corresponds to the species M. cochillia, and a PCR assay was designed for its specific diagnosis. The association of huge cockle mortality with M. cochillia infection urges extreme caution to avoid spreading this disease.

The spatio-temporal distribution of the mesoparasitic copepod Pennella sp. on the short-finned squid Illex coindetii was studied monthly during 1993 in the southeastern North Atlantic (Galician waters, NW Spain). A total of 600 individuals were analysed separately considering two areas (north and west) with different hydrographical characteristics. Six pennellid stages were found: a copepodite, four chalimus stages and the free-living adult male and female. Epidemiology was assessed using the parasite demographic parameters. The results revealed a marked seasonal pattern in the number of parasites in squid samples from the northern area. This seasonal pattern was not detected in squids from the western area. The observed frequency distributions of parasites on the host population were aggregative at each sampling area. Infection values were extraordinarily high when they are compared with published host-parasite records for other representatives of the genus Pennella. Advantages of the parasite aggregation pattern and possible explanations of the seasonality variations in the number of parasites observed in squid samples from the northern area are discussed.

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.

This paper examines the species composition of the parasite fauna and the values of infection for seven species of cephalopods in the Mediterranean at the Tyrrhenian Sea (West coast of Italy). Results suggest the important role of cephalopods as intermediate hosts in the life cycle of anisakine nematodes and pennellid copepods. The low host specificity (i. e., eurixenous condition) of metazoan parasites in cephalopods worldwide is also reinforced., No disponible

10 páginas, 4 figuras, 1 tabla, Long-tem (1991-1991) information on parasitic infection by anisakid nematodes in cephalopods and top predators (marine mammals) of the south-eastem area of the North Atlantic underlines the important role of imail cetaceans as final hosts for A. simplex. The ommastrephid squid Illex coindetii, Todaropsis eblanae and Todrtrodes sagittatus are the most important cephalopod paratenic hosts in the life cycle of the parasite. Information on parasite flow and parasite-caused diseases could be of use for stock assessment pulposes. Moreover, it largely agrees with what is known about interactions between prey (squid) and predator (cetacean) in the same area. Parasitic castration and stomach wall ulceration were the most impofiant parasitecaused effects recorded in infected cephalopods and cetaceans respectively., Financial assistance was provided by the Consellería de Educación e Ordenación Universitaria, Xunta de Galicia, under the project XUGA3O1I0A97

3 páginas, 2 figuras., A developmental stage of a species of eimeriorin Apicomplexa was found heavily infecting the kidney of the European cormorant Phalacrocorax aristotelis aristotelis beached on the coast of Galicia after the oil spill from the ‘Prestige’. The immature condition of the parasite made a precise taxonomic identification impossible. The infection caused kidney damage as a result of pressure atrophy and mechanical displacement exerted by oocysts which occupied large areas of host tissue. A cellular host reaction was always apparent in the kidney of the infected cormorants. Although the oil effect is assumed to be responsible for the death of the cormorants, the impact of the coccidiosis should be monitored in the European shag populations.

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

10 páginas, 3 figuras, 1 tabla, Public organizations operating in health and food-safety sectors are increasingly realizing the advantages of the long-term view of risk uncertainties associated to biological hazards, served-up in the short-term to anticipate the problem and its handling. Thus, the horizon scanning is becoming a major strand in proactive risk management and patient-consumer protection continuity. This approach was recently explained in the scientific opinion on risk assessment of parasites in fishery products by the European Food Safety Authority, EFSA (2010), followed by the launching of a funding scheme for a specific EU Framework Program Project under the Knowledge Based Bio-Economy concept, KBBE (FP7-KBBE-2012-6), which drives the new EU 2020 strategy. The aim of this paper is to examine horizon scanning issues in relation to public health and industrial concern on the presence of parasites in fishery products recorded in the Rapid Alert System for Food and Feed (RASFF) System. We focus on specific threats, targets, methods and challenges as a means of acquiring management goals and future objectives. The proposed horizon scanning identifies emerging ideas/technologies for an early handling of parasitized fish stocks/products for priority setting to inform strategic planning of stakeholders, policy-makers and health services. In order to accomplish this, a set of risk GIS maps illustrating the state of art about the presence of the zoonotic Anisakis spp. on commercial fish stocks of the last 65 years was firstly developed. Secondly, a program of 108 surveys among fish sellers of Galicia (NW Spain) were carried out with the main objective of getting information about hazard recognition, fish product management practices, quality self-controls and corrective and preventive measures in use. Additionally, during the “I International Symposium on strategies for management of parasitized seafood products” (Vigo, Spain), groups of researchers, technologists, official inspectors and industries participated in round tables with 3 different perspectives: market-industry, inspection and academia. All scanners agreed that the status quo to manage fish parasites in the production-to-consumption food pathway is unsatisfactory. The central message proposed a stable network performance based on collaborative software to provide multi-level information for industrial management of parasite contaminants in fish products. The discussion group also proposed to invigorate collaborative translational research and professional training as key drivers to fuel technological innovations and tech transfer, which may help to minimize/eliminate the risk of parasites that have public health and economic impacts in fish products., We thank Xunta de Galicia for financial support under Projects Parcode (10TAL033E), Anitech (10TAL001CT) and IN841C. Authors would like to thanks to Miguel Bao (ECOBIOMAR) the excellent bibliographic work, and to Carlos Vello/Luis Outeiriño (Comercial Hospitalaria Grupo 3 S.L.) and Rosa Fernández (CETMAR) for their help in the organization of the I International Symposium on strategies for management of parasitized seafood products. M. Llarena-Reino and M. Regueira thanks Fundação para a Ciência e a Tecnologia and European Social Fund for financial support under grants SFRH/BD/45398/2008 and SFRH/BD/51038/2010 respectively.

8 páginas, 2 figuras, 2 tablas, A total of 982 individuals distributed in 11 lots belonging to 10 fish species from three Atlantic FAO fishing areas were sampled and examined to detect the presence of anisakid larvae in fish muscle. After hazard identification by genetic sequencing and exposure assessment by anatomic extent and demographic characterization of infection, all data were fitted for each fish species to a new proposed scoring schema of parasite prediction. In the absence of a criterion standard method for inspection and precise definition of the quantum satis for parasites in contaminated fish lots, the inspection rating scheme called SADE (Site of infection, Assurance of quality, Demography, Epidemiology) may help fish industries to precisely handle and to evaluate the likely outcome of infected fish lots after being diagnosed. For this purpose, a supporting flow diagram for decision was defined and suggested. This new performance assessment tool has the aim of staging fish lots, thus helping in planning manufacture, commercial, and research decisions during self-management programs. This novel scoring system provides an improved inspection format by implementing the occurrence stratification for parasites to guide Hazard Analysis and Critical Control Points (HACCP) programs for the uniform exchange of information among fish industries, administration and researchers, thus facilitating standardization and communication. In the future, this scoring version could be validated (in terms of classification and wording) for similar overall predictive purposes in other muscular parasites infecting seafood products., Xunta de Galicia, Projects INCITE-07MMA015CT, IN841C, 10TAL001CT, and 10TAL033E, Fundaçao para a Ciencia e a Tecnologia, Grant SFRH/BD/45398/2008

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.

12 páginas, 3 figuras, 3 tablas, 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., This research was supported by CAIBEX (CTM-2007- 66408-CO2-01), LARECO (CTM-2011-25929), CGL2007- 63221, and CGL2012-39545. M.G. was funded by the Junta para la Ampliación de Estudios Predoctorales (CSIC) cofinanced with Fondo Social Europeo (ESP) funds

This paper represents an updated review of the published and unpublished records of parasites collected from cephalopod molluscs (teuthoid squids, cuttlefish and octopods) in waters off Spain, with special mention for those inhabiting coastal and shelf waters off Galicia (NW Spain). The examination of about 1600 cephalopods in the southeastern North Atlantic Ocean revealed a new parasite species Stellicola hochbergi , 37 new host records for Atlanto-Iberian waters, and 18 new host records for the world Ocean (three for coccidian Aggregatidae; three and one for cestodes Phyllobothriidae and Tentaculariidae; five and one for nematodes Anisakidae and Cystidicolidae; one and four for copepods Lichomolgidae and Pennellidae). Results suggests the important role of cephalopods as intermediate or final hosts in the life cycles of ten systematic groups of parasites.

11 páginas, 3 figuras, 3 tablas, 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., This research was conducted within the National Projects ‘CAIBEX’ (CTM-2007-66408-CO2-01), LARECO (CTM 2011-25929) and was also supported by project CGL2007- 63221, MEC, Spain. The equipment was partially funded with FEDER Funds. M.G. was supported by a JAE-CSIC predoctoral scholarship.

Disseminated neoplasia (DN), an oyster disease resembling leukaemia, has been reported in a number of species of marine bivalve molluscs. The disease is characterised by a proliferation of abnormal circulating cells of unknown origin resulting in the invasion of tissues and organs, frequently with a fatal end of the affected individuals. To obtain a more comprehensive view of bivalve cancer processes, suppressive subtracted hybridisation (SSH) and quantitative RT-PCR (q-PCR) approaches were combined to investigate changes in the transcriptome of Ostrea edulis haemolymph cells associated to DN. Two SSH libraries were constructed and 587 expressed sequence tags (ESTs) were sequenced, obtaining 329 ESTs which showed expression changes in neoplastic process. Transcription expression analyses (q-PCR) were done for a total of 24 genes that could be relevant in neoplastic process, including genes with role in the regulation of cell cycle, apoptosis or chromosomal defects. Most of those genes had not been reported in association with cancer in non-vertebrate organisms. The over-expression and under-expression of some of those genes in DN-affected oysters was in agreement with observations in vertebrate cancer. The results herein reported contribute to cancer understanding in bivalve molluscs.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C gigas. On December 2003, naive and Bonamia-relatively resistant flat oysters from Ireland. Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O-2(-)] and hydrogen peroxide [H2O2]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreae in vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded. (C) 2012 Elsevier Inc. All rights reserved.

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 ± 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 ± 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease This study was supported by the Ministerio de Ciencia e Innovación of the Spanish government, under the project AGL2006-11809. B.G.P. and R.V. were supported by an Isidro Parga Pondal research fellowship from Xunta de Galicia (Spain); E.A. was supported by a Ramón y Cajal contract of the Ministerio de Ciencia e Innovación of the Spanish Government SI

Biological and habitat characterization of the non-indigenous invasive species Xenostrobus securis was undertaken in the Ria de Vigo. This study included genetic identification of mussel samples collected from introduced and endemic areas, and the assessment of mussel size, population abundance, geographic distribution, pathological condition, and sediment composition of substrata type. The mussel had a marked patchy distribution, being more abundant in brackish sites with fine sediments and high organic matter content. Pathological analysis revealed that X. securis does not play any role as vector for introducing allochthonous pathogens in the Ria de Vigo. Nevertheless, depending on its invasiveness potential, the mussel could be a key host favouring spreading and epizootic outbreaks of marteliosis which is known to be harmful for local bivalve populations. Phylogenetic analyses of the COI gene placed all the resulting sequences in a clade within the genus Xenostrobus and its phylogeny congruent with an Australian/Pacific origin. The COI tree suggests two historical introductions in European waters. One of these invasions seems to have started in Galicia, moving from there towards Italy and France, while the geographical spread of the second invasion cannot be deciphered, although the Australian/Pacific origin of this invasion seems very possible. The 18S network is congruent with one invasion starting in Galicia or in Italy, as the Australian haplotype is closely related to the haplotype found in these areas. Several hypotheses accounting for the colonization history of this species in Galician waters are discussed.

7 páginas, 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.

Long-term (1990-1996) results of recording gastric ulcers in the common dolphin are presented for the NW Spanish Atlantic coast. The occurrence and abundance of cetacean carcasses (belonging to 10 species) with gastric ulcers are also given and discussed. Ulcerations were detected in 17.2% of the animals examined, with 15% for common dolphins and 0 to 29.4% in other cetacean species. A positive relation was noted between ulcer counts and length and maturity of Delphinus delphis. Clusters of the nematode Anisakis simplex could be seen embedded in the gastric ulcers of 3 common dolphins. It could be concluded that gastric ulcers are non-fatal lesions in cetaceans stranded in NW Spain.

7 pages, 10 figures.-- The PDF this article is free., Numerous ribbon-like aggregations of eggs of a trematode didymozoid were found embedded in the connective tissue, mostly between the epoxial skeletal muscle fibres, of mature mackerel Scomber scombrus collected from post-recruits of the eastern stock. Egg masses contained numerous developmental stages, including pre-vitellogenous oocytes to fully embryonated eggs. Genomic DNA isolation, PCR amplification, cloning, and sequencing strongly supported the view that the eggs belong to a yet unidentified didymozoid. There was minor muscular damage to the host, namely focal distortion of host fibre architecture to accommodate the parasite, and the mild host response consisted of different stages of encapsulation. Concurrent heavy infection makes the mackerel flesh unappealing to eat, although the quality of the fish is not significantly affected and there is no public health danger., This work was supported by Marie Curie Fellowship contract No. QLK5-CT2000-52101 under the Fifth Framework Programme of the European Community.

9 páginas, 4 figuras, 1 tabla, The interferon regulatory factor (IRF) family comprises transcription factors that regulate the expression of interferon and interferon-related cytokines. Using the RACE technique, we have determined the complete cDNA sequence of turbot (Scophthalmus maximus) and sea bream (Sparus aurata) IRFs. These sequences shared characteristics with other IRFs of fish, mammals and birds, and showed high similarity with IRF-1. Indeed, they were included in the IRF-1 cluster of the phylogenetic tree constructed with IRF-1 and IRF-2 sequences of several organisms, and presented a low number of basic amino acid residues in the carboxy-terminal end of the proteins. All of these characteristics led to the identification of turbot and sea bream IRFs as IRF-1. Two IRF-1 sequences were obtained for both turbot and sea bream, and we named them turbot/sea bream IRF-1a and IRF-1b. Turbot IRF-1a differed from turbot IRF-1b in four nucleotides. The presence of both IRF types in cDNA from 45 turbot livers was determined by RFLP, suggesting the duplication of the gene. Sea bream IRF-1b presented a deletion of 121 bp in its ORF compared to sea bream IRF-1a, and since both IRF types were present in all 25 cDNAs analyzed by PCR, we hypothesized that the truncated sea bream IRF-1b was probably an alternative splicing product. Turbot and sea bream IRF-1 expression was constitutive in every analyzed organ, as reported before for other fish species. Poly I:C significantly stimulated turbot IRF-1 expression in muscle, spleen and kidney 24 h post-treatment, while viral haemorrhagic septicemia virus (VHSV) induced a differential expression of this factor in kidney 8 h after infection. These results do not agree with those previously reported for flounder and trout IRF. Other expression experiments with turbot leukocytes stimulated in vitro with poly I:C and with brain and kidney of sea bream infected with nodavirus did not bring out differential IRF expression levels in stimulated samples with respect to controls., This research has been supported by the Ministerio de Ciencia y Tecnolog´ıa under project BIO2001 2324-C02, and also by a Marie Curie Fellowship of the European Community Fifth Framework Programme under contract number QLK5-CT-2002-51499.