Characterization of Aliivibrio fischeri strains associated with disease outbreak in brill Scophthalmus rhombus.

Three bacterial isolates were recovered from a disease outbreak with high mortality affecting brill Scophthalmus rhombus (Linnaeus, 1758). Moribund fish showed no external signs of disease, but plentiful haemorrhages were observed in liver. On the basis of phenotypic and genotypic characterization, the isolates were identified as Aliivibrio fischeri. The phenotypic profile of the isolates was basically similar to that of the type strain of this species, although some discrepancies were observed, mainly in the BIOLOG GN profile. The main cellular fatty acids of strain a591 were also consistent with this species. The highest 16S rDNA sequence similarities were recorded with the type strain of A. fischeri (99.07%); other Aliivibrio species showed similarity values below 96%. The highest sequence similarities with gyrB, rpoD and recA genes were also recorded with A. fischeri type strain (99.31, 98.99 and 95.29% similarity, respectively). DNA-DNA hybridization assays confirmed that these isolates belong to A. fischeri; levels of DNA relatedness were 73.5 to 86.2% with isolate a591 (reciprocal values of 86.9 to 99.04%). Finally, a virulence evaluation of the isolates using Senegalese sole fry was also performed; significant mortalities (100% mortality within 5 d) were recorded by intraperitoneal injection, but only with high doses of bacteria (2 × 106 cfu g-1 body weight).

Isolation of Lacinutrix venerupis strains associated with disease outbreaks in sea bream Sparus aurata and European sea bass Dicentrarchus labrax.

Four Gram-negative bacterial isolates were recovered from 2 disease outbreaks that occurred in 2013 affecting European sea bass Dicentrarchus labrax fry and sea bream Sparus aurata adults. Main symptoms were erratic swimming, eroded fins and, in the sea bream outbreak, haemorrhages on the body surface; bacteria were always recovered from internal organs, almost in pure culture. On the basis of phenotypic characterization and 16S rRNA gene sequence analysis, the isolates were identified as Lacinutrix venerupis, a bacterium not previously reported as a fish pathogen. The highest 16S rDNA sequence similarities were recorded with the type strain of this species (99.9-100% similarity), while other species showed similarities below 97%, the closest relative being L. mariniflava (96.3% similarity). Phenotypic characterization showed some discrepancies with the L. venerupis type strain (mainly in BIOLOG GN profile); however, DNA-DNA hybridization assays with L. venerupis and L. mariniflava type strains confirmed that these isolates belong to the former species (levels of DNA relatedness were 98-100% and 38-50%, respectively). Finally, a virulence evaluation of the isolates using Senegalese sole Solea senegalensis fry was also performed; significant mortalities (80-100% mortality within 4 d) were recorded after intraperitoneal injection, but only with high doses of bacteria (107colony forming units fish-1). Further studies will be necessary to determine the importance of this species as a fish pathogen.

Ostreid herpesvirus in wild oysters from the Huelva coast (SW Spain).

This is the first report of ostreid herpesvirus 1 microvariant (OsHV-1 µVar) infecting natural oyster beds located in Huelva (SW Spain). The virus was detected in 3 oyster species present in the intertidal zone: Crassostrea gigas (Thunberg, 1793), C. angulata (Lamarck, 1819) and, for the first time, in Ostrea stentina Payraudeau, 1826. Oysters were identified by a specific polymerase chain reaction (PCR) and posterior restriction fragment length polymorphism (RFLP) analysis based on cytochrome oxidase I (COI) mitochondrial DNA. Results confirmed that C. angulata still remains the dominant oyster population in SW Spain despite the introduction of C. gigas for cultivation in the late 1970s, and its subsequent naturalization. C. angulata shows a higher haplotype diversity than C. gigas. OsHV-1 virus was detected by PCR with C2/C6 pair primers. Posterior RFLP analyses with the restriction enzyme MfeI were done in order to reveal the OsHV-1 µVar. Detections were confirmed by DNA sequencing, and infections were evidenced by in situ hybridization in C. gigas, C. angulata and O. stentina samples. The prevalence was similar among the 3 oyster species but varied between sampling locations, being higher in areas with greater harvesting activities. OsHV-1 µVar accounted for 93% of all OsHV-1 detected.

Characterization of Vibrio tapetis strains isolated from diseased cultured Wedge sole (Dicologoglossa cuneata Moreau).

The first isolation of Vibrio tapetis from Wedge sole (Dicologoglossa cuneata) is reported. The bacterium was recovered from ulcers of ailing cultured fish, from two different outbreaks occurred in spring 2005. The four isolates found (a200, a201, a204 and a255) were biochemically, genetically and serologically characterized and diagnosis was confirmed by PCR V. tapetis specific primers and multilocus sequencing analysis (MLSA). The isolates constituted a homogeneous phenotypic and genotypic group, being distinct to the already serological and genetic groups defined within the species. A virulence evaluation of the isolate a255 was also carried out; however this strain was unable to induce disease in fry and juvenile Wedge sole.

In vivo genotoxicity and stress defences in three flatfish species exposed to CuSO4.

We have used the comet assay to analyse, after 3h, 24h and 6 days, the genotoxic effect in vivo of applying a single intraperitoneal injection of CuSO4, at a concentration of 2mg/kg, to adult specimens of Solea senegalensis, Dicologlossa cuneata and Scophthalmus rhombus. Metals content (Cu, Zn and Cd) in liver was also measured. The activity of key stress defences was evaluated by analysing antioxidant enzyme activity (catalase (CAT), superoxide dismutase (SOD), total glutathione peroxidase (t-GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH)), metallothionein (MT) and heat shock proteins (HSP70 and HSP60). The results show that CuSO4 intake generates high and cumulative levels of genotoxicity throughout the 6 days in all 3 species. After 6 days, metals content detected in specimens showed significant differences from controls. Inter-species differences were detected in enzyme activity (P<0.05). A clear response to CuSO4 was detected only in S. rhombus, with an increase of MT and a decrease of HSPs. Variations in antioxidant defence levels and their comparative responses to the stress-inducing agent are discussed.

First isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata (Moreau).

The first isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata, is reported. The pathogen was recovered from ulcers of cultured fish, from three different outbreaks. The six isolates obtained were biochemically and serologically characterized and diagnosis was confirmed by polymerase chain reaction using specific primers and partial 16S rRNA gene sequencing. The isolates constituted a homogeneous phenotypic group; however, they belong to two of the different serotypes described within this species. A virulence evaluation of the isolates using Wedge sole fry was also performed.

The centromeric satellite of the wedge sole (Dicologoglossa cuneata, Pleuronectiformes) is composed mainly of a sequence motif conserved in other vertebrate centromeric DNAs.

Here, a new satellite-DNA family is isolated and characterized from wedge sole, Dicologoglossa cuneata Moreau, 1881 (Pleuronectiformes), a fish having a small genome. This satellite-DNA family of sequences was isolated by conventional cloning after digestion of genomic DNA with the DraI restriction enzyme. Repeat units are 171 bp in length with a high AT content (63%). Several runs of consecutive adenines and thymines were found, and concomitantly computer analyses revealed that these regions are prone to acquire stable sequence-directed curvature. Especially remarkable is that the DraI sequences are composed almost entirely of the repetition of up to fourteen 9-bp motifs (T/C)GTC(A/C)AAAA similar to other vertebrate centromeric satellite-DNA sequences. In fact, we demonstrate the origin of this satellite through duplication of this motif plus the addition of a stretch of cytosines. The centromeric location and the presence in this satellite-DNA sequence of not only different vertebrate motifs (CENP-B box, pJalpha) but also others such as the CDEIII motif of Saccharomyces cerevisiae reveal a possible role in centromere function. All these characteristics provide important information on the origin, function, and the evolution of the centromeric satellite DNAs in wedge sole.

Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral.

Metal accumulation and some of their biochemical effects have been studied in oysters (Crassostrea angulata) and mussels (Mytilus galloprovincialis) of the South Atlantic Spanish littoral. Especial attention has been paid to antioxidant defences and oxidative damage to biomolecules. Deep differences in the response of oysters and mussels to metal pollution were found. Oysters, with the higher metal loads of both species, showed increased antioxidant defences, and less extensive oxidative damage. In contrast, mussels, which accumulated much lower metal concentrations, showed clear increases in oxidized biomolecules, in agreement with their low increases in the antioxidant defence mechanisms. Our results suggest that mussels are more sensitive and less well adapted to metal pollution, probably explaining their absence in the most contaminated studied site, Mazagón. We conclude that oysters can be used as more sensitive bioindicator of pollution in the South Spanish littoral, and as a suitable model to study the adaptation to metal pollution.

The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence.

Marteilia refringens is a paramyxean parasite which infects the flat oyster Ostrea edulis and mussels (Mytilus galloprovincialis), where it has been attributed to a separate species, Marteilia maurini, by several authors. Doubts persist though as to the existence or not of two species of Marteilia in Europe. We have devised a molecular method for the diagnosis of M. refringens based on 358 bp nested-PCR of the rDNA intergene spacer (rDNA IGS) which is capable of detecting 0.5 fg of M. refringens DNA. Molecular characterization of this spacer indicates that the Marteilia parasites which infect oysters and mussels are two different strains of the same species.

Molecular characterization of the ribosomal RNA gene region of Perkinsus atlanticus: its use in phylogenetic analysis and as a target for a molecular diagnosis.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S-5S-18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.

Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants.

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

Promutagen activation by fish liver as a biomarker of littoral pollution.

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.

Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts.

Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.