A Genome-Wide Association Study for Host Resistance to Ostreid Herpesvirus in Pacific Oysters (Crassostrea gigas).

Ostreid herpesvirus (OsHV) can cause mass mortality events in Pacific oyster aquaculture. While various factors impact on the severity of outbreaks, it is clear that genetic resistance of the host is an important determinant of mortality levels. This raises the possibility of selective breeding strategies to improve the genetic resistance of farmed oyster stocks, thereby contributing to disease control. Traditional selective breeding can be augmented by use of genetic markers, either via marker-assisted or genomic selection. The aim of the current study was to investigate the genetic architecture of resistance to OsHV in Pacific oyster, to identify genomic regions containing putative resistance genes, and to inform the use of genomics to enhance efforts to breed for resistance. To achieve this, a population of ∼1,000 juvenile oysters were experimentally challenged with a virulent form of OsHV, with samples taken from mortalities and survivors for genotyping and qPCR measurement of viral load. The samples were genotyped using a recently-developed SNP array, and the genotype data were used to reconstruct the pedigree. Using these pedigree and genotype data, the first high density linkage map was constructed for Pacific oyster, containing 20,353 SNPs mapped to the ten pairs of chromosomes. Genetic parameters for resistance to OsHV were estimated, indicating a significant but low heritability for the binary trait of survival and also for viral load measures (h2 0.12 - 0.25). A genome-wide association study highlighted a region of linkage group 6 containing a significant QTL affecting host resistance. These results are an important step toward identification of genes underlying resistance to OsHV in oyster, and a step toward applying genomic data to enhance selective breeding for disease resistance in oyster aquaculture.

Genotoxic effects of CdS quantum dots and Ag2S nanoparticles in fish cell lines (RTG-2).

The increasing use of nanotechnologies will lead to significant releases of engineered nanoparticles into the aquatic environment, where their impact is still poorly characterized. In the present paper, the genotoxic and cytotoxic properties of CdS quantum dots (QDs) and silver sulphide (Ag2S) coated with methyl polyethylene glycol (M-PEG) were investigated in a rainbow trout cell line (RTG-2). The results showed that CdS QDs were highly cytotoxic at high concentrations (10 and 50µg/ml), and exhibited a concentration-dependent genotoxicity in the sub-toxic range (0.01-1µg/ml) after 24h exposure. Ag2S showed neither genotoxic nor cytotoxic effects.

Ocean acidification increases the toxicity of contaminated sediments.

Ocean acidification (OA) may alter the behaviour of sediment-bound metals, modifying their bioavailability and thus toxicity. We provide the first experimental test of this hypothesis with the amphipod Corophium volutator. Amphipods were exposed to two test sediments, one with relatively high metals concentrations (Σmetals 239 mg kg(-1) ) and a reference sediment with lower contamination (Σmetals 82 mg kg(-1) ) under conditions that mimic current and projected conditions of OA (390-1140 µatm pCO2 ). Survival and DNA damage was measured in the amphipods, whereas the flux of labile metals was measured in the sediment and water column (WC) using Diffusive Gradients in Thin-films. The contaminated sediments became more acutely toxic to C. volutator under elevated pCO2 (1140 µatm). There was also a 2.7-fold increase in DNA damage in amphipods exposed to the contaminated sediment at 750 µatm pCO2 , as well as increased DNA damage in organisms exposed to the reference sediment, but only at 1140 µatm pCO2 . The projected pCO2 concentrations increased the flux of nickel and zinc to labile states in the WC and pore water. However, the increase in metal flux at elevated pCO2 was equal between the reference and contaminated sediments or, occasionally, greater from reference sediments. Hence, the toxicological interaction between OA and contaminants could not be explained by e ffects of pH on metal speciation. We propose that the additive physiological effects of OA and contaminants will be more important than changes in metal speciation in determining the responses of benthos to contaminated sediments under OA. Our data demonstrate clear potential for near-future OA to increase the susceptibility of benthic ecosystems to contaminants. Environmental policy should consider contaminants within the context of changing environmental conditions. Specifically, sediment metals guidelines may need to be reevaluated to afford appropriate environmental protection under future conditions of OA.

Field surveys reveal the presence of anti-androgens in an effluent-receiving river using stickleback-specific biomarkers.

This study was designed to assess whether the removal of endocrine disrupting chemicals (EDCs) and other substances from a Waste Water Treatment Works (WWTW) effluent (receiving water: R. Ray, Swindon, UK) by granular activated carbon (GAC) affected biomarkers of exposure to EDCs [vitellogenin (VTG) and spiggin] in male and female three-spined sticklebacks in the receiving water. A nearby river (R. Ock), with a negligible effluent loading, was used as a control. On each river fish were sampled from four sites on five occasions both before and after remediation of the WWTW effluent. The results show for the first time in a UK field study a clear seasonality of blood VTG concentrations in wild male fish, following closely the VTG profile in female fish from both rivers. VTG levels in male fish from the R. Ray were significantly reduced after the GAC installation. However, VTG levels in males from the control sites also varied significantly across the same period, reducing the significance of this finding. A laboratory exposure to oestradiol (using site-specific lower and upper levels of oestrogenic activity) failed to elevate VTG concentrations in male sticklebacks suggesting that concentrations in the effluent, even prior to remediation, may not have exceeded a critical sensitivity threshold. Most importantly, a significant increase in female kidney spiggin content (a highly specific biomarker of xeno-androgen exposure) occurred in fish in the R. Ray after the GAC installation to levels comparable with those in fish from the control river. The significance of this finding is strengthened by the fact that during the pre-remediation period in the R. Ray, female spiggin levels increased with increasing distance from the WWTW. Our results provide the first in vivo evidence of the presence of anti-androgens in a UK WWTW effluent. To our knowledge this is the first UK-based comprehensive field study on the effects of a WWTW upgrade on biomarkers of EDC exposure using a sentinel fish species and our findings confirm the value of the stickleback as a model species for studying EDCs both in the laboratory and in the wild.

Hepatic transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol.

An established three-spined stickleback (Gasterosteus aculeatus) cDNA array was expanded to 14,496 probes with the addition of hepatic clones derived from subtractive and normalized libraries from control males and males exposed to model toxicants. Microarrays and one-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, together with individual protein and gene biomarkers were employed to investigate the hepatic responses of the stickleback to ethinyl-estradiol (EE(2)) exposure. Male fish were exposed via the water to EE(2), including environmentally relevant concentrations (0.1-100ng/l) for 4 days, and hepatic transcript and metabolite profiles, kidney spiggin protein and serum vitellogenin concentrations were determined in comparison to controls. EE(2) exposure did not significantly affect spiggin concentration but significantly induced serum vitellogenin protein at the threshold concentration of 32ng/l. (1)H NMR coupled with robust univariate testing revealed only limited changes, but these did support the predicted modulation of the amino acid profile by transcriptomics. Transcriptional induction was found for hepatic vitellogenins and choriogenins as expected, together with a range of other EE(2)-responsive genes. Choriogenins showed the more sensitive responses with statistically significant induction at 10ng/l. Real-time polymerase chain reaction (PCR) confirmed transcriptional induction of these genes. Phosvitinless vitellogenin C transcripts were highly expressed and represent a major form of the egg yolk precursors, and this is in contrast to other fish species where it is a minor component of vitellogenic transcripts. Differences in inducibility between the vitellogenins and choriogenins appear to be in accordance with the sequential formation of chorion and yolk during oogenesis in fish.

The organophosphorous pesticide, fenitrothion, acts as an anti-androgen and alters reproductive behavior of the male three-spined stickleback, Gasterosteus aculeatus.

Fenitrothion (FN) is a widely used organophosphorous pesticide that has structural similarities with the clinical anti-androgen flutamide. The potential for FN to act as an anti-androgen (at exposures of 1, 50, and 200 microg FN/l over a 26-day period) was assessed in male three-spined sticklebacks, Gasterosteus aculeatus, by measuring kidney spiggin concentration, nest-building, and courtship behavior. Spiggin is the glue protein that male sticklebacks use to build their nests and is directly controlled by androgens. FN exposure significantly reduced spiggin production as well as nest-building activity. It also adversely affected courtship--especially the 'zigzag dance' and biting behavior of the males. FN thus appears to have anti-androgenic effects on both the physiology and behavior of the male stickleback.

Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.

Intercalibration exercise using a stickleback endocrine disrupter screening assay.

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17beta-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17beta-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Larval Development and Vitellin-like Protein Expression in Palaemon elegans Larvae Following Xeno-oestrogen Exposure.

Certain anthropogenic chemicals, most notably xeno-oestrogens, are known to have the potential to disrupt vertebrate endocrine systems. For example, induction of the female-specific protein, vitellogenin, in male fish is a well-known effect of exposure to xeno-oestrogens and serves as a biomarker of such exposure. There have been few comparable studies of putative biomarkers of endocrine disruption in invertebrates. An exception is the upregulation of vitellin-like larval storage protein (LSP) expression in the barnacle cypris larva following exposure to oestrogenic chemicals. The current study aimed to establish whether larvae of the glass prawn, Palaemon elegans, are likewise susceptible to xeno-oestrogen exposure. Using a polyclonal antiserum to P. elegans apolipovitellin, an 86 kDa polypeptide was detected by western blotting in the larval and early postlarval stages of this species. An indirect ELISA applied to the soluble protein fraction of larval homogenates determined that the titre of this putative LSP ranged, depending on larval stage, from 0.48-0.67 ng µg(-1). Exposure of P. elegans larvae to the xeno-oestrogen 4-n-nonylphenol (4-NP), at 0.2-20 µg L(-1), resulted in a significant, concentration-independent increase in putative LSP levels of 5-18%. Conversely, exposure to the natural oestrogen, 17ß-oestradiol (E(2)), at 0.2 and 20 µg L(-1), led to a significant concentration-independent decline (up to 11%) in LSP levels. Whether the effect of 4-NP results from endocrine disruption is not known, however, an oestrogen receptor-mediated effect is unlikely. Other than a slight increase in larval mortality when exposed to 4-NP at 2 µg L(-1), neither 4-NP nor E(2) significantly affected development, growth or survival of P. elegans larvae.