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Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

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AIMS: This study aims to assess the use of marine lactic acid bacteria (LAB) to reduce Vibrio parahaemolyticus levels during oyster depuration process. METHODS AND RESULTS: The inhibitory effect of 30 marine LAB strains against V. parahaemolyticus strains was evaluated by in vitro assays. A total of three positive strains (Latilactobacillus sakei SF1583, Lactococcus lactis SF1945, and Vagococcus fluvialis CD264) were selected for V. parahaemolyticus levels reduction during oyster depuration. Pacific oysters Crassostrea gigas were artificially and independently contaminated by four GFP-labelled V. parahaemolyticus strains (IFVp201, IFVp69, IFVp195, and LMG2850T) at 105 CFU ml-1 and then exposed by balneation to 106 CFU ml-1 of each LAB strains during 24 h, at 19°C. Quantification of V. parahaemolyticus in haemolymph by flow cytometry revealed variations in natural depuration of the different V. parahaemolyticus strains alone. Furthermore, the addition of LABs improved up to 1-log bacteria ml-1 the reduction of IFVp201 concentration in comparison to the control condition. CONCLUSIONS: Although further optimizations of procedure are needed, addition of marine LABs during oyster depuration may be an interesting strategy to reduce V. parahaemolyticus levels in Crassostrea gigas.

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Recent advances in glycobiotechnology show that bacterial exopolysaccharides (EPS) presenting glycosaminoglycan (GAG)-like properties can provide a valuable source of bio-active macromolecules for industrial applications. The HE800 EPS, named diabolican, is a marine-derived anionic high-molecular-weight polysaccharide produced by Vibrio diabolicus CNCM I-1629 which displays original structural features close to those of hyaluronic acid. We investigated the impact of carbon and nitrogen substrates on both Vibrio diabolicus growth and diabolican production. Both substrates were screened by a one-factor-at-a-time method, and experimental designs were used to study the effect of glucose, mannitol, and ammonium acetate various concentrations. Results showed that the medium composition affected not only the bacterium growth and EPS yield, but also the EPS molecular weight (MW). EPS yields of 563 and 330 mg L-1 were obtained in the presence of 69.3 g L-1 glucose and 24.6 g L-1 mannitol, respectively, both for 116.6 mM ammonium acetate. MW was the highest, with 69.3 g L-1 glucose and 101.9 mM ammonium acetate (2.3 × 106 g mol-1). In parallel, the bacterial maximum specific growth rate was higher when both carbon and nitrogen substrate concentrations were low. This work paves the way for the optimization of marine exopolysaccharide production of great interest in the fields of human health and cosmetics.

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Sex-determining modes remain unknown in numerous species, notably in fishes, in which a variety of modalities have been reported. Additionally, noninvasive individual sexing is problematic for species without external sex attributes or for early life stages, requiring cytogenetic or molecular analyses when sex chromosomes or sex-linked markers have been characterized. Genomics now provide a means to achieve this. Here, we review common sex-determination systems and corresponding statistical methods for identifying sex-linked genetic markers and their use for sex assignment, focusing on single nucleotide polymorphism (SNP) markers derived from reduced representation sequencing methods. We demonstrate the dependence of expected sex assignment error on the number of sex-linked SNPs and minor allele frequency. The application of three methods was made here: (a) identification of heterozygote excess in one sex, (b) FST outlier analysis between the two sexes and (c) neuronal net modelling. These methods were applied to a large SNP data set (4604 SNPs) for 1680 thornback rays (Raja clavata). Using method (a), nineteen putative sex-linked SNPs were identified. Comparison with the reference genome of a related species (Amblyraja radiata) indicated that all 19 SNPs are probably located on the same chromosome. These results suggest that thornback ray has a XX/XY sex-determination system. Method (b) identified eight SNPs probably located on different chromosomes. Method (a) led to the lowest sex assignment error among the three methods (4.2% error for females and 3.7% for males).

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Cryptosporidium, a zoonotic pathogen, is able to infect a wide range of hosts including wild and domestic animals, and humans. Although it is well known that some parasites are both fish pathogens and recognized agents of zoonosis with a public health impact, little information is available concerning the prevalence of Cryptosporidium in wild aquatic environments. To evaluate the prevalence of Cryptosporidium spp. in commercially important edible marine fish in different European seas (English channel, North sea, Bay of Biscay, Celtic sea and Mediterranean sea), 1,853 specimens were collected as part of two surveys. Nested PCR followed by sequence analysis at the 18S rRNA gene locus was used to identify Cryptosporidium spp. The overall prevalence of Cryptosporidium spp. in sampled fish reached 2.3% (35 out of 1,508) in a first campaign and 3.2% (11 out of 345) in a second campaign. Sequence and phylogenetic analysis of positive samples identified Cryptosporidium parvum (n = 10) and seven genotypes which exhibited between 7.3 and 10.1% genetic distance from C. molnari, with the exception of one genotype which exhibited only 0.5-0.7% genetic distance from C. molnari. Among 31 analyzed fish species, 11 (35.5%) were identified as potential hosts for Cryptosporidium. A higher prevalence of Cryptosporidium spp. was observed in larger fish, in fish collected during the spring-summer period, and in those caught in the North East Atlantic. Pollachius virens (saithe) was the most frequently Cryptosporidium positive species. In fish infected by other parasites, the risk of being Cryptosporidium positive increased 10-fold (OR: 9.95, CI: 2.32-40.01.04, P = 0.0002). Four gp60 subtypes were detected among the C. parvum positive samples: IIaA13G1R1, IIaA15G2R1, IIaA17G2R1, and IIaA18G3R1. These C. parvum subtypes have been previously detected in terrestrial mammals and may constitute an additional source of infection for other animals and in particular for humans. Microscopical examination of histological sections confirmed the presence of round bodies suggestive of the development of C. parvum within digestive glands. We report herein the first epidemiological and molecular data concerning the detection of Cryptosporidium in edible marine fish in European seas surrounding France broadening its host range and uncovering potential novel infection routes.

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Tuna fisheries and processing represent economic activities of paramount importance around the world. Most of these products are traded for human consumption and in general are highly demanded commodities. However, not all tuna products achieve the same market price, some consumers are willing to pay a huge amount of money for certain species (i.e. Japanese market for Bluefin tuna) while other species are rather affordable (i.e. Skipjack tuna), therefore mislabelling has been observed frequently. We collected and analysed 545 tuna samples in six European countries, including fresh, frozen and canned products, and we have investigated whether or not these products were correctly labelled under European and national legislations. We found an overall mislabelling rate of 6.79%; in particular, 6.70% of the fresh and frozen tuna products and 7.84% of canned tuna were mislabelled, and only in the case of fresh and frozen tuna samples significant differences among countries were found. Mislabelling rates for Atlantic Bluefin tuna labelled products were very high, ranging from 50 up to 100%. In general, mislabelling was higher when specific names were included in the labels. The "tuna" umbrella term is a very popular one with consumers, but also one that remains vulnerable to ambiguity, hampering efforts towards market transparency and with potential negative consequences to the adequate management of tuna species stocks.

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OBJECTIVES: The eps locus in Vibrio diabolicus is involved in the production of the biotechnologically valuable HE800 EPS. In this study, the distribution and diversity of similar eps gene clusters across Vibrionaceae and its variability in relation to phylogenetic relationship were investigated. The aim was to provide a better knowledge of the eps gene cluster importance and to facilitate discovery of new EPS with potent interesting bioactivities. RESULTS: Seventy percent of the 103 genome sequences examined display such an eps locus with a high level of synteny. However, genetic divergence was found inside some monophyletic clades or even between some strains of the same species. It includes gene insertions, truncations, and deletions. Comparative analysis also reveals some variations in glycosyltransferase and export systems genes. Phylogenetic analysis of the Vibrionaceae eps gene clusters within Vibrionaceae suggests a vertical transfer by speciation but also pinpoints rearrangement events independent of the speciation.

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Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

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The sea lamprey Petromyzon marinus, which is among the most phylogenetically ancient vertebrates, is a hematophagous ectoparasite that feeds on vertebrates and is considered vulnerable in Europe but is a pest in the North American Great Lakes. We conducted a literature review of helminth parasites of P. marinus and investigated postmetamorphic lampreys sampled in rivers and northeast Atlantic coastal waters (western France) during spawning migration. Based on the literature review, 16 helminth taxa have been recorded in P. marinus, among them 14 in North America but only 2 in Europe, with no species in common between these areas. Specific parasites are lacking, and helminth parasites recorded in P. marinus are mostly opportunistic and are trophically transmitted to fish hosts with both extremely low prevalence and mean intensity. Thus, P. marinus seems an unusual host that is probably infected through accidental ingestion of parasites by microphagous larvae (ammocoetes) and/or hematophagous postmetamorphs. Our field study supports this hypothesis, since only a single third-stage larva of Anisakis simplex sensu stricto was found in 2 postmetamorphic P. marinus among the 115 individuals dissected. This opportunistic, trophically transmitted, and cosmopolitan nematode species has never been recorded in North American sea lampreys and only once in Galician rivers (southern Europe). Infestation pathways of P. marinus by A. simplex are proposed vis-à-vis the feeding strategy of postmetamorphs and fish host species which potentially harbor anisakid larvae in their musculature. More generally, the complexity of biotic interactions is discussed considering P. marinus both as a host for helminth parasites and as a parasite for hosts such as fish and mammals, which are also potential predators of sea lamprey.

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Overlapping external morphometric characters easily confound the flatfishes Solea aegyptiaca and Solea solea (Soleidae) in areas of the Mediterranean Sea where both species live in sympatry. This leads to uncertainties in the fisheries and marketing of the species, in addition to misinterpretations in biogeography and conservation studies. This paper describes a simple restriction fragment length-based diagnostic test that differentiates S. solea from S. aegyptiaca, as well as from other species of the Soleidae family. Furthermore, the two species living in sympatry in the Gulf of Kavala (North Aegean Sea, Greece) present significant qualitative differences in muscle fatty acid composition, a property that can also be used to distinguish the two cryptic species.

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Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

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Traceability in the fish food sector plays an increasingly important role for consumer protection and confidence building. This is reflected by the introduction of legislation and rules covering traceability on national and international levels. Although traceability through labeling is well established and supported by respective regulations, monitoring and enforcement of these rules are still hampered by the lack of efficient diagnostic tools. We describe protocols using a direct sequencing method based on 212-274-bp diagnostic sequences derived from species-specific mitochondria DNA cytochrome b, 16S rRNA, and cytochrome oxidase subunit I sequences which can efficiently be applied to unambiguously determine even closely related fish species in processed food products labeled "anchovy". Traceability of anchovy-labeled products is supported by the public online database AnchovyID ( http://anchovyid.jrc.ec.europa.eu), which provided data obtained during our study and tools for analytical purposes.

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Postmortem tenderization is caused by enzymatic degradation of key structural proteins in myofibrils as well as in extracellular matrix, and of proteins involved in intermyofibrillar linkages and linkages between myofibrils and the sarcolemma. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicate that calpains and cathepsins may be responsible for degradation of these proteins. Other phenomena occurring in cells postmortem (pH drop, sarcoplasmic Ca2+ increase, osmotic pressure rise, oxidative processes) may act in synergy with proteases. Our understanding of the underlying mechanisms of muscle degradation should be improved for an accurate evaluation of the postmortem muscle changes and consequently of the fish quality.

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High-pressure processing is a nonthermal technique ensuring food product safety and enabling a longer shelf life. The purpose of this experiment was to evaluate the effect of high pressure on the main proteolytic enzymes involved in fish muscle degradation during storage. Enzymes were extracted with sarcoplasmic proteins from Dicentrarchus labrax sea bass white muscle. Activity of cathepsins B, D, H, and L was quantified in protein extract, whereas calpain activity was evaluated after isolation from its endogenous inhibitor. High-pressure processing up to 500 MPa enhanced the activity of cathepsin B, H, and L, whereas the activity of cathepsin D increased up to 300 MPa and decreased above 300 MPa. With regard to calpain activity, high-pressure processing led to a decrease of activity, which was zero above 400 MPa. We suggest a leading explanation based on simultaneous deactivation of enzymes and an increase of liberation from lysosomes for cathepsins and on dissociation of subunits for calpains.

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A direct sequencing method based on a 103 bp diagnostic sequence derived from a species-specific mitochondrial DNA cytochrome b sequence of 150 bp obtained by Polymerase Chain Reaction was tested for the identification of 47 commercial canned sardine and sardine-type products from various countries. Multiple alignment of 14 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned sardine (

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A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24 degrees C in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required.