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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. • HTS data remarks the high dependence aquafeed manufacturing industry has on forage species. • Hysterothylacium aduncum and Phocanema krabbei were identified in 8 out of ten analysed feeds. • Genomic results complement previous works, as HTS revealed allergenic nematodes occurring in fish feed marketed in Spain. • The above finding is of highly concern regarding dietetic recommendations to sensitized patients to anisakids. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • Visible parasites in the swordfish are causing a growing sanitary and economic issue. • 214 pennellids from 167 Xiphias gladius were subjected to diagnosis and epidemiology. • Pennellid cephalothoraxes' and infected swordfish slices were morphologically studied. • All the cephalothoraxes were morphologically identified as Pennella instructa. • Proactive self-inspections must ensure safer and high quality standard fish products. Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 121bp 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 24h post-treatment, while viral haemorrhagic septicemia virus (VHSV) induced a differential expression of this factor in kidney 8h 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The nematodes of the genus Anisakis are among the most prevalent parasites found in fishes and marine mammals and they are the main cause of human anisakiasis. Genetic studies have described two species, A. simplex and A. pegreffii, in European waters parasitizing several hosts, being the European hake, by far, the fish with higher infection values. The aim of this study is to enhance the knowledge of the distribution and population structure of the Anisakis species infecting hakes from the major European fishing areas through the use of genetic analysis. This study provides useful information about the genetic diversity of these parasites in the different fishing areas and fish tissues, valuable findings to understand the parasite speciation to different hake tissues and how they are structured along European waters. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The euphausiidNyctiphanes couchiiand an unidentified mysid have been found, for the first time, with third-stage larvae (L3) of theAnisakis simplexcomplex in the mesozooplanktonic community of the coastal upwelling system in Galicia (NW Spain). Parasite larvae were molecularly identified using the internal transcribed spacer (ITS) region. The prevalence of these parasites in the euphausiid population was 0.0019%. The existence of parasites in a variety of mesozooplankton organisms suggests that the transmission routes ofA. simplexsensu stricto andA. pegrefiiare wider than expected. The results suggest that these twoAnisakisspecies are not specific to their intermediate hosts. Finally, the recruitment ofA. simplexcomplex may be affected by oceanography, differing under upwelling or downwelling conditions. [ABSTRACT FROM PUBLISHER]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small (∼22 nt) 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cloning and characterisation of genes involved into disseminated neoplasia in oyster. [•] Phylogenetic relationships of each gene were characterised. [•] Gene expression is modulated by bonamiosis, disseminated neoplasia, and both. [•] Occurrence of both diseases in the oyster intensifies changes in gene expression. [ABSTRACT FROM AUTHOR]

Abstract: Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes. [Copyright &y& Elsevier]

Abstract: 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, naïve 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 [] 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]