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The use of wild small fish species as feed for aquaculture has clearly an economic incentive by speeding the growth of farmed species. Since feed ingredients are sourced from wild fisheries the farmed species could contain natural contaminants which may introduce food safety concerns. In this study, we used High-Throughput Sequencing (HTS) to explore the whole DNA profile of ten dry commercial feeds commonly used by Spanish fish farming companies. The feeds were mainly made of species within the genus Sprattus, Ammodytes and Clupea, and vegetables of the genus Triticum. In the feeds, DNA sequences of parasitic nematodes of fishes (˂1 % total OTUs) were also identified. A taxonomic assignment of query sequences, using a phylogeny-based approach, estimation of pairwise nucleotide identities within and between sequence groups and haplotype network analysis, allow assign short query sequences to the species Phocanema krabbei (Anisakidae) and Hysterothylacium aduncum (Rhaphidascarididae). Both species were identified as ingredient in two and six fish feeds, respectively. This result is of highly concern regarding dietetic recommendations to sensitized patients to anisakids, considering the growing evidence on the possible allergenic potential of both genera, and the recent data on the transfer of anisakid heat-resistant allergens from fishmeal to farm and aquaculture animals

This paper compiles the data regarding the first occurrence of Oithona davisae and O. atlantica in NW Spain, which is supported by morphological and molecular analysis. Additionally, we investigated the seasonal dynamics of the invasive O. davisae, revealing that its abundance is conditioned by upwelling-downwelling patterns in the Rías Baixas of Galicia. Temperature was the most correlated factor, with higher abundances in upwelling relaxation-downwelling events. More studies in long-term zooplankton dynamics and molecular analysis are needed to determine if O. davisae is displacing other native species of the same genus, such as O. atlantica, in Galician waters.

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

The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments

[Statement of the problem] Evidence of Anisakidae larvae in a high and rising number of fish species of commercial interest around the world has remarkably increased. The importance of fish infection by Anisakidae lies in their repercussion on seafood safety and quality, increasing awareness about anisakids as a re-emergent risk in fish-eating consumers. Parasite Biobank was implemented at the UTB-CSIC in 2013. Until now, it managed more than 100000 parasite samples (mainly Anisakidae) from almost 25000 extractions. Furthermore, during that period a BoB (EpiData, software Bio-o-Bank Vitro®) was generated, with all the traced information for any extraction and sample. Those data showed to be an invaluable asset for evaluating and monitoring the Anisakidae presence in fisheries and aquaculture systems, but also for other significant objectives, such as developing risk management tools. From 2018 to 2022, as part of several collaborative projects between CSIC and the Spanish Seafood industry, a technological support task was designed to develop new strategies for anisakid risk management. Fish industry operators made the inspection and banked the results. As data was the banked “golden” items, we developed a quality control process to ensure their quality and traceability, [Proposed solution] Data collection: Detection method is a key step. Operators evaluated the presence of zoonotic Anisakidae following the UV-Press inspection method, as described in ISO 23036-1:2021. Operators were trained at the UTB, which also provided mentoring, advising and technical assistance regarding the inspection process as well as the data traceability within the Biobank platform tool. Verification plan: In order to ensure that the inspection method was properly implemented according to ISO 23036-1:2021 standard, pressed portions of fish, randomly selected and previously inspected by operators, were sent to UTB (i.e. verifier), where they were re-examined by highly specialised laboratory staff., [Validation] As UTB act as verifier and their results were stablish as the “gold standard”, we designed an internal quality control to validate our own performance on the ISO 23036-1:2021., [Conclusion] Data quality is a measure of the condition of data based on factors such as accuracy, completeness, consistency and reliability. We (UTB-CSIC) developed a normalized protocol to routinely assess on these parameters of quality for banked data provided by registers (fish inspectors) during collection processes.

[Planteamiento del problema] Existe una amplia diversidad de muestras biológicas, tanto de origen animal (peces, zooplancton, moluscos, etc), vegetal (algas, fitoplancton…) o ambiental (sedimentos o agua) que se utilizan en actividades de investigación y en diferentes sectores que evalúan y gestionan los diferentes servicios ecosistémicos del medio marino. Asociadas a estas muestras se identifican vulnerabilidades/riesgos relacionados con la presencia de organismos patógenos (ej., parásitos, virus, hongos y bacterias), responsables tanto de importantes pérdidas económicas a los sectores productivos, como de enfermedades zoonóticas de interés en salud pública. Actualmente, este tipo de riesgos biológicos intrínsecos a la obtención, manipulación y gestión de las muestras, no se han tenido en cuenta. En el marco de la estrategia “One Health” planteada como referente en los planes de bioseguridad del IIM-CSIC, se pone de manifiesto la necesidad de identificar los riesgos biológicos presentes en las muestras de origen marino gestionadas desde los laboratorios para poder evaluarlos y, así, emprender acciones preventivas que aseguren una correcta seguridad e higiene de los trabajadores., [Solución planteada] El primer objetivo de esta estrategia se centró en las muestras de agua marina, que constituye el hábitat común a todos los organismos marinos y es el vector o reservorio de gran número de patógenos. El objetivo fue realizar, mediante metabarcoding de ADN ambiental (eDNA), la identificación molecular de parásitos protozoarios y metazoarios, hongos y bacterias presentes en aguas que prestan diferentes servicios ecosistémicos. Esta identificación nos permitirá conocer la identidad de los agentes biológicos presentes en cada tipo de agua y evaluar y categorizar su riesgo., [Conclusiones] Este estudio permitirá la identificación de numerosos agentes biológicos, lo cual nos permitirá abordar el desarrollo de protocolos en materia de bioseguridad, prevención de riesgos laborales y gestión de residuos. Todo ello, puede ser de utilidad para establecer medidas adecuadas que aseguren una correcta seguridad e higiene, tanto en centros de investigación marina como en empresas vinculadas al complejo mar industrial

[Statement of the problem] Marine samples take part in an ecosystem which includes, not only animals, plants and their environment, but also other organisms such as parasites, viruses, bacteria and fungi. Many of these microscopic organisms are pathogens for marine flora and fauna, causing significant economic losses in the productive sector. Moreover, an important number of them are zoonotic pathogens which are spread through food, water and the environment. Thus, as stated in the One Health approach (World Health Organization) there are a clear link between animal health, human health and environment. It is therefore essential a holistic vision aimed to prevent, detect, contain and minimize the potential public and animal health risks. However, the marine biological risks are therefore not taken into account in sampling, handling and analysis of marine samples by research centers and private companies. Additional efforts need to be undertaken to identify zoonotic pathogens from marine samples, carry out the risk assessment and preventive actions in order to ensure workers safety and hygiene., [Proposed solution] The Biobank Technical Unit (UTBCSIC) is a traceable high-quality sampling platform, which include marine organisms, their DNAs and associated data. Nowadays, the priority objective of UTB-CSIC is to contribute to identify and assess on marine biological risks. Thus, during the last year we focused on generating an environmental DNA (eDNA) Biobank from marine water that provides different ecosystem services. As a first approximation to evaluate the genetic diversity and identify potential zoonotic pathogens, DNA metabarcoding libraries for parasites, bacteria and fungi were constructed and sequenced in Illumina PE250 platform. Bioinformatic analysis will allow to know the identity of microorganisms included in each type of water and categorize and assess the risk for each biological agent. Standardized procedures and protocols will be developed, including preventive measures for handling of each type of water., [Conclusions] Biobanked eDNA will be useful for researching of wide variety of organisms and for deepening both in studies of animal and human health, as well as, in food safety. This information will be essential to assess the risk of manipulating marine samples in research centers, and to carry out preventive actions in order to ensure workers safety.

Fish are an important part of a healthy, well-balanced diet. They provide a good source of protein and vitamins and are a primary dietary source of heart-healthy omega-3 fatty acids. While eating fish has nutritional benefits, it also has potential risks. Fish can take in harmful biohazards from naturally exploited ecosystems and the food they eat. Regarding parasite hazards, there is plenty of scientific evidence related to the raised natural impact of marine parasites on the 3S stands for Fisheries: Security, Safety, and Sustainability. Many parasites, which occur in exploited ecosystems, could potentially overcome a hierarchical series of barriers to cause spillover effects on wild fish productivity and sustainability, and/or seafood consumer´s health. The conclusion behind an intensive decade of fish parasite monitoring at ICES fishing areas tell us about quality product rejection with significant economic losses along the fish-production value chain, serious parasite risk exposure for fish-eating consumers, unsustainable fishing practices on heavily-parasitized stocks still being considered, and an increased confused concern at the fish industry on how managing these challenges. The idea that parasite biodiversity may constitutes a threat to efficient ICES fish production value chains provokes a very real sound perspective of parasites being integrated within the fish holobiont paradigm, and as a part of the socioecological management system in any particular fishery. We provide data to argue that a parasite hazard warning system based on the 3S model (from net to plate), integrated in an adaptive co-management of fisheries, could be the best choice highlighted by the cost-effectiveness to prevent such socioecological potentially damaging impact of parasites to ICES fisheries. Overall, we propose a design for major parasite threats integrated in the One Health approach needed in ecosystem management practices

[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.

The Fish PARASITE Biobank (FPB) was construct­ed on 2013 as a traceable high-quality sampling platform far the largest surveillance plan ever conducted in EU fish production value chains. The Biobank collects and records fish zoonotic parasite samples and their associated information. Since 2016, the FPB is certified with the quality management system standard ISO 9001. One distinguishable promising future challenge far the FPB is to provide high-quality genomic DNA (gDNA) stored under strict conditions of traceability. The different species of zoonotic fish parasites must be iden­tified by molecular methods, thérefore, gDNA included in the Biobank can be su ita ble far the development of DNA bar­codes, DNA-signature sequences that will allow an accurate identification of these pathogens. Nowadays, few barcoding studies are available about zoonotic marine fish parasites. Here, we establish a suitable procedure to obtain high-quality DNA of zoonotic parasites and review the barcode sequences disposable in public databases far them

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

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.

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

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 schem

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.

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 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.

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 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.

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 in

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.

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.

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 susceptibi

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.

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

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.

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.

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 t

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.

Biological monitoring refers to the use of living organisms to evaluate environmental conditions. Of particular relevance to the health of marine ecosystems is the improvement of methodologies of biological monitoring to provide highly ecologically sensitive indices of exposure. We have shown that anisakid nematodes, a parasite group widely distributed in oceans that infects a wide range of host species, can accumulate essential and non-essential metals to levels far in excess of their host tissues. The fact that they could be used as biomarkers of trace-metal contamination in studies of environmental impact suggests a potential use as a monitor species in a marine ecosystem under anthropogenic stress because they might produce a warning or alert signal of high ecological relevance.

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.

The cloning and sequencing of the small subunit (SSU) ribosomal DNA gene from Kudoa rosenbuschi (myxosporean species associated with post-mortem myoliquefaction process in the Argentinean hake Merluccius hubbsi) is reported. The SSU rDNA was found to contain 1740 bp with a single polymorphic site with either a C or T at position 221. The sequence data obtained in this study and those known sequences of Kudoa species deposited in the GenBank were all analyzed to construct a phylogenetic tree. Nucleotide sequences showed the highest degree of identity with K. funduli, followed by K. miniauriculata, K. clupeidae and K. dianae. Phylogenetic analysis placed K. rosenbuschi in the same branch of K. clupeidae and K. funduli, and showed it to be closely related to K. dianae, K. paniformis and K. miniauriculata.

We analyse the cephalopod–parasite systems taking into account the abiotic and biotic factors that may affect the host risk of infection (contact) with parasites. On the basis of 2000 individuals comprising 10 cephalopod species collected at a microgeographic area (Galician waters, NW Spain), an attempt is made to find associations between parasite relative species diversity (RSD) and cephalopod life cycle characteristics. Additionally, an index of similarity and the ratios of adult/immature (A/I) and specialist/generalist (S/G) regarding the parasite condition were used to define cephalopod ecological groupings at those species with similar risk of becoming infected with a given parasite fauna. Results show the existence of three ecological groupings (coastal, intermediate, and nerito-oceanic), which suggest that the ecological niche of a cephalopod species is more important in determining its risk of parasitic infection than is phylogeny.

We analyse the cephalopod–parasite systems taking into account the abiotic and biotic factors that may affect the host risk of infection (contact) with parasites. On the basis of 2000 individuals comprising 10 cephalopod species collected at a microgeographic area (Galician waters, NW Spain), an attempt is made to find associations between parasite relative species diversity (RSD) and cephalopod life cycle characteristics. Additionally, an index of similarity and the ratios of adult/immature (A/I) and specialist/generalist (S/G) regarding the parasite condition were used to define cephalopod ecological groupings at those species with similar risk of becoming infected with a given parasite fauna. Results show the existence of three ecological groupings (coastal, intermediate, and nerito-oceanic), which suggest that the ecological niche of acephalopod species is more important in determining its risk of parasitic infection than is phylogeny

Gamogony and sporogony of Aggregata octopiana were commonly observed during histological examination of the digestive tract of wild Octopus vulgaris from Ría 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.

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.