INTRODUCTION: all decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. METHODS: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. RESULTS: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. DISCUSSION: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Aquaculture is the fastest-growing farmed food sector and will soon become the primary source of fish and shellfish for human diets. In contrast to crop and livestock production, aquaculture production is derived from numerous, exceptionally diverse species that are typically in the early stages of domestication. Genetic improvement of production traits via well-designed, managed breeding programmes has great potential to help meet the rising seafood demand driven by human population growth. Supported by continuous advances in sequencing and bioinformatics, genomics is increasingly being applied across the broad range of aquaculture species and at all stages of the domestication process to optimize selective breeding. In the future, combining genomic selection with biotechnological innovations, such as genome editing and surrogate broodstock technologies, may further expedite genetic improvement in aquaculture.

Hypoxia is one of the major threats to biodiversity in aquatic systems. The association of hypoxia with nutrient-rich effluent input into aquatic systems results in scenarios where hypoxic waters could be contaminated with a wide range of chemicals, including metals. Despite this, little is known about the ability of fish to respond to hypoxia when exposures occur in the presence of environmental toxicants. We address this knowledge gap by investigating the effects of exposures to different levels of oxygen in the presence or absence of copper using the three-spined sticklebacks (Gasterosteus aculeatus) model. Fish were exposed to different air saturations (AS; 100%, 75% and 50%) in combination with copper (20 μg/L) over a 4 day period. The critical oxygen level (Pcrit), an indicator of acute hypoxia tolerance, was 54.64 ± 2.51% AS under control conditions, and 36.21 ± 2.14% when fish were chronically exposed to hypoxia (50% AS) for 4 days, revealing the ability of fish to acclimate to low oxygen conditions. Importantly, the additional exposure to copper (20 μg/L) prevented this improvement in Pcrit, impairing hypoxia acclimation. In addition, an increase in ventilation rate was observed for combined copper and hypoxia exposure, compared to the single stressors or the controls. Interestingly, in the groups exposed to copper, a large increase in variation in the measured Pcrit was observed between individuals, both under normoxic and hypoxic conditions. This variation, if observed in wild populations, may lead to selection for a tolerant phenotype and alterations in the gene pool of the populations, with consequences for their sustainability. Our findings provide strong evidence that copper reduces the capacity of fish to respond to hypoxia by preventing acclimation and will inform predictions of the consequences of global increases of hypoxia in water systems affected by other pollutants worldwide.

Survival of marine fishes that are exposed to elevated near-future CO2 levels is threatened by their altered responses to sensory cues. Here we demonstrate a physiological and molecular mechanism in the olfactory system that helps to explain altered behaviour under elevated CO2. We combine electrophysiology measurements and transcriptomics with behavioural experiments to investigate how elevated CO2 affects the olfactory system of European sea bass (Dicentrarchus labrax). When exposed to elevated CO2 (approximately 1,000 µatm), fish must be up to 42% closer to an odour source for detection, compared with current CO2 levels (around 400 µatm), decreasing their chances of detecting food or predators. Compromised olfaction correlated with the suppression of the transcription of genes involved in synaptic strength, cell excitability and wiring of the olfactory system in response to sustained exposure to elevated CO2 levels. Our findings complement the previously proposed impairment of γ-aminobutyric acid receptors, and indicate that both the olfactory system and central brain function are compromised by elevated CO2 levels.

AbstractFor non-kin cooperation to be maintained, individuals need to respond adaptively to the cooperative behaviour of their social partners. Currently, however, little is known about the biological responses of individuals to experiencing cooperation. Here, we quantify the neuroregulatory response of Trinidadian guppies (Poecilia reticulata) experiencing cooperation or defection by examining the transcriptional response of the oxytocin gene (oxt; also known as isotocin), which has been implicated in cooperative decision-making. We exposed wild-caught females to social environments where partners either cooperated or defected during predator inspection, or to a control (non-predator inspection) context, and quantified the relative transcription of the oxt gene. We tested an experimental group, originating from a site where individuals are under high predation threat and have previous experience of large aquatic predators (HP), and a control group, where individuals are under low predation threat and naïve to large aquatic predators (LP). In HP, but not LP, fish brain mid-section oxt relative transcription varied depending on social partner behaviour. HP fish experiencing cooperation during predator inspection had lower oxt transcription than those experiencing defection. This effect was not present in the control population or in the control context, where the behaviour of social partners did not affect oxt transcription. Our findings provide insight into the neuromodulation underpinning behavioural responses to social experiences, and ultimately to the proximate mechanisms underlying social decision-making.

Microsporidiosis poses an ongoing global threat to human health and animal wellbeing, in immunocompromised populations. As obligate intracellular parasites, microsporidia possess a greatly reduced genome and metabolome, whilst exhibiting an extensive array of extracellular proteins for host invasion and adherence.
The aim of the work described in this thesis is to provide a clearer understanding of the microsporidian infectious pathway and presents a three- step pipeline to aid the discovery of novel proteins and mechanisms within that infectious pathway. Throughout the study, a combination of in-silico and laboratory-based investigations were performed to place equal emphasis on the proteins of the parasite and their respective hosts. Firstly, an in-silico experiment was devised using hidden Markov modelling and orthology-based prediction to collate and expand current putative microsporidian spore wall and polar tube proteins. Next, a novel proteomic “SHIPP” method was designed to isolate and identify novel microsporidian surface proteins and their respective host target sites. Lastly, two transfection methods of microsporidia were attempted to validate spore-host interactions: namely, trial use of cell penetrating peptides; and an adaptation of the CHoP-In mutagenesis approach for the knockdown of host membrane proteins.
The results produced a consensus database comprising 294 surface protein orthologues, split across 34 distinct protein types. As such it represents one of the most comprehensive databases of its kind, to date. A trial of the “SHIPP” assay was able to successfully select for the extracellular proteins of both microsporidia and host cells, whilst identifying six novel protein candidates in microsporidia for further characterisation. Finally, use of cell penetrating peptides was shown to be an ineffective method for microsporidian transfection, whilst the adaption of the CHoP-In protocol for the mutagenesis of host cells requires further work. CHoP-In components were successfully designed, but could not be fully implemented into a complete trial run. Ultimately, it is believed that the components of this pipeline will assist in the discovery of novel microsporidian surface proteins, with future adaptations potentially assisting with the design of novel microsporidian treatments and the generation of resistant livestock to minimise the impacts of microsporidiosis.

INTRODUCTION: all decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. METHODS: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. RESULTS: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. DISCUSSION: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Freshwater ecosystems are affected by pollution from many sources, impacting the biodiversity within. Exposures can be chronic, testing an organism’s defence mechanisms over time, or intermittent, spiking in severity at point sources or as the seasons change. Despite these real-world scenarios being common, experimental studies often do not consider intermittent, repeated exposures within their designs. This could lead to a misinterpretation of safe concentrations within environments if these intermittent exposure conditions result in alterations of tolerance in the exposed organism. It is also important to understand how the life stage of the individual at the time of the exposures may impact their response, to both the initial and subsequent exposures. In this thesis, I set out to understand how chemical exposures during periods of sensitivity during development alter the responses to exposures at later stages, using zebrafish (Danio rerio) as a model species.
I hypothesised that the first four hours of zebrafish development (one-cell stage to sphere stage) would be more sensitive to environmental stressors than exposures that are initiated after this point, due to developmental processes occurring during this time window including epigenetic reprogramming, zygotic genome activation and changes in chorion permeability. To test this, I used two pre-exposure time periods, one that included this proposed period of sensitivity (initiated at one-cell stage) and another initiating at four hours post fertilisation (sphere stage). I also proposed that the developmental stage directly after hatching would be a sensitive period due to the loss of the chorion membrane allowing for increased interactions with toxic chemicals within the exposure medium. Therefore, I used this period as the subsequent re-exposure period.
To investigate these hypotheses, I used metals as the chemical stressors, specifically copper, silver and mercury. All are environmentally relevant as they are found to pollute aquatic environments around the globe, and their toxicity to fish has been studied, albeit rarely under these intermittent exposure scenarios. Silver can utilise some of the same uptake and cellular transport mechanisms as copper, however copper is an essential micronutrient whilst silver has no biological function, so comparisons of zebrafish embryo response were explored. Mercury, specifically methylmercury (due to the organic form’s ability to bioaccumulate), was also used in this thesis as the metal has been shown to cause epigenetic alterations, has no biological function and is a widespread pollutant in many tropical environments.
Pre-exposure to copper, regardless of whether that pre-exposure period encompassed the proposed period of sensitivity, increased the larval tolerance to re-exposure compared to naïve individuals. However, pre-exposure encompassing the hypothesised period of sensitivity was significantly more sensitive to copper during embryogenesis than pre-exposures initiated at four hours post fertilisation. The increased tolerance to re-exposure was associated with copper-induced delayed hatching, and increased tolerance was no longer observed when exposures were initiated in hatched larvae. The mechanisms responsible for the delay in hatching were explored, and hatching enzyme production was not shown to be disrupted. Addition of water containing hatching enzyme caused premature hatching in control treatment groups, whilst not initiating hatching in those pre-exposed. Therefore, I proposed that hatching enzyme binding sites on the chorion surface were affected by the copper exposures, resulting in reduced efficiency of the enzyme in causing chorion breakdown.
Silver pre-exposures did not alter tolerance to re-exposure regardless of the pre-exposure period, despite the earlier exposure period that encompassed our proposed period of sensitivity being significantly more sensitive to silver exposure than the later pre-exposure period. The gene expression of several genes involved in the response to metal exposures and in epigenetic pathways were investigated at 48 hours post fertilisation, to determine if pre-exposure during either period altered gene regulation 24 hours after the exposure. No significant differences in gene expression were reported, which could have been due to the depuration period allowing gene expression to return to normal levels, or due to insufficient concentration of silver during the embryonic exposure.
Zebrafish pre-exposed to methylmercury showed no significant mortality compared to controls and increased tolerance to subsequent exposures, principally lower mortality rates if the pre-exposure period encompassed the proposed period of sensitivity. Transcriptome profiles were investigated to understand how gene regulation differed between treatment groups and whether pre-exposure during epigenetic reprogramming had caused significant, long term alterations to gene regulation. In groups not re-exposed at larval stage, transcriptomes were very similar between controls and those pre-exposed, indicating as with the silver study that any potential gene expression alterations caused by the initial exposure return to baseline levels during depuration periods for these two metals. Upon re-exposure, those pre-exposed during embryogenesis showed significantly lower alterations of gene transcription, with pathways involved in apoptosis, immune response and DNA repair being less over/under–represented compared to naïve fish exposed to methylmercury for the first time. Naïve treatment groups, and to a lesser extent treatment groups pre-exposed to methylmercury during the later development stage, also showed suppression of pathways involved in nervous system and eye development, whilst zebrafish pre-exposed during the proposed sensitivity period did not. These results indicated that pre-exposure had decreased the impact of methylmercury exposure on gene regulation which could explain the decreased mortality observed. Differences in gene expression related to histones modification were also observed in all groups, suggesting that epigenetic alterations occur as a result of methylmercury exposure. I propose that these results provide support for a potential epigenetic mechanism associated with more efficient response to methylmercury during re-exposure and resulting in the increase in tolerance observed.
My research highlights the importance of considering intermittent exposures in environmental risk assessment and conservation efforts, especially when determining the tolerance of fish species to pollutants. I also highlight the importance of initiating exposures as early in development as possible, to capture potential periods of increased sensitivity and to represent more closely real-world scenarios. This is especially prudent when exposures during this time can have more profound effects on embryos and/or affect their tolerance to subsequent exposures.

Amoebiasis, is a parasitic disease caused by Entamoeba histolytica, causing a wide range of clinical manifestations, from asymptomatic infection to amoebic dysentery and liver abscess, and encystation is a key process enabling this parasite to cause disease. Because encystation of E. histolytica has not been successfully reproduced under laboratory conditions, it is often conducted in surrogate model species. The cyst wall of E. invadens, a model for E. histolytica, is mainly composed of chitin fibrils, two chitin-binding lectins, Jacob that cross-link chitin fibrils and Jessie that self-aggregates on the cyst wall and an enzyme, chitinase, which remodels chitin. Current research aims to identify new biological drugs that can target the cyst formation process but that does not affect human cells. In this study I aimed to study the membrane dynamics of E. invadens during encystation using Langmuir trough apparatus and cell fluctuation analysis. The effect of cyst wall components; chitin and its deacetylated form chitosan on E. invadens plasma membrane lipids (PML) and red blood cells (RBCs) membrane have been studied. I demonstrated that the addition of chitin and chitosan to E. invadens PML has increased the stiffness of plasma membrane lipids and increased the rigidity of RBCs membrane. Furthermore, I aimed to study the effect of Jacob and Jessie lectin on PML and RBCs membrane. Jacob and Jessie were successfully amplified and cloned into an expression vector, however, several experiments to purify these proteins were unsuccessful. This may be due to the protein being unstable and/or toxic to E. coli host, or caused by inefficient transcription, translation and/or posttranslational modifications. In order to resolve these issues, it may be necessary to use different host cells such as mammalian or insect cells. Together, the data presented in this thesis provides evidence that chitin and chitosan contribute to increased rigidity of the lipid monolayer/bilayer. This knowledge in the future may contribute to research aimed at the development of treatments to combat amoebiasis.

Globally, aquaculture development is leading the way in the ‘Blue Revolution’ with it rapidly outstripping wild-capture fisheries. However, in the face of climate change and greenhouse gas emissions, it is vital that going forward, aquaculture develops in a sustainable and ecologically aware manner. One aspect of this is ensuring farms are operating at maximum efficiency, particularly from a biological perspective. Here we present an assessment of the impact that elevated environmental CO2 often found in fish farms can have on the digestive physiology of salmonids and the development of new tools for the field of fish physiology.
Chapter 2 presents an experimental investigation into how exposure to elevated CO2 interacts with the metabolic costs of digestion. Utilising unsealed intermittent flow respirometry it was observed that rainbow trout (Oncorhynchus mykiss) are metabolically robust to elevations in environmental CO2 across the scope of one large meal and 6 small meals. This indicates that the reduction in growth observed in salmonids exposed to elevated CO2 is not due to an increased metabolic cost of digestion and therefore may be due to other aspects of a fish’s energy budget.
The contents of Chapter 3 validate a novel method of fish phlebotomy that opens up a range of possibilities for assessing physiological responses that have thus far been unattainable with current methods. By comparing the novel method to grab ‘n’ stab and cannulation it was found that the novel method was able to avoid the acute stress responses associated with grab ‘n’ stab and generate data comparable with cannulation. This method particularly allows assessment of accurate acid-base responses to stimuli in a more ‘natural’ state and in fish smaller than cannulation can typically achieve. Notably, this allows for fish to be voluntarily feeding before blood samples are taken, opening up routes for novel methods of assessing physiological responses to feeding.
Utilising the method developed in Chapter 3, Chapter 4 assesses the internal physiological processes associated with feeding and how these processes are impacted by exposure to elevated CO2. Utilising a combination of post-prandial acid-base whole animal flux measurements, and characterisation of post-prandial blood acid-base state, haematology, and osmotic components, a full characterisation of the physiological processes of digestion were assessed. This chapter found significant alterations to typical transport processes associated with feeding and indicates that elevated CO2 may cause switching of the routes of gastric bicarbonate excretion. There was also evidence of elevated CO2 impacting protein utilisation and assimilation supporting the evidence presented in Chapter 2.

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-β signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout.

White Spot Syndrome Virus (WSSV) causes White Spot Disease (WSD) and is historically the most devastating disease in the shrimp industry. Global losses from this disease have previously exceeded $3 bn annually, having a major impact on a global industry worth US$19 bn per annum. Shrimp are cultured predominantly in enclosed ponds that are subject to considerable fluctuations in abiotic conditions and WSD outbreaks are increasingly linked to periods of extreme weather, which may cause major fluctuations in pond culture conditions. Combined with the intensity of production in these systems, the resulting suboptimal physicochemical conditions have a major bearing on the susceptibility of shrimp to infection and disease. Current knowledge indicates that pond temperature and salinity are major factors determining outbreak severity. WSSV appears to be most virulent in water temperatures between 25 and 28 °C and salinities far removed from the isoosmotic point of shrimp. Elevated temperatures (>30 °C) may protect against WSD, depending on the stage of infection, however the mechanisms mediating this effect have not been well established. Other factors relating to water quality that may play key roles in determining outbreak severity include dissolved oxygen concentration, nitrogenous compound concentration, partial pressure of carbon dioxide and pH, but data on their impacts on WSSV susceptibility in cultured shrimps is scarce. This illustrates a major research gap in our understanding of the influence of environmental conditions on disease. For example, it is not clear whether temperature manipulations can be used effectively to prevent or mitigate WSD in cultured shrimp. Therefore, developing our understanding of the impact of environmental conditions on shrimp susceptibility to WSSV may provide insight for WSD mitigation when, even after decades of research, there is no effective practical prophylaxis or treatment.

Wildlife are exposed to a variety of environmental stressors, including toxic chemicals, which often occur intermittently and over long periods of time, driven by fluctuations in discharge and environmental conditions. Survival of wildlife populations is therefore critically dependent upon their ability to adapt to repeated chemical exposures. However, current chemical testing methods for environmental risk assessment generally consider only a single exposure of a previously naïve population of animals, failing to account for altered responses of organisms depending upon their exposure history. Evidence suggests that organisms may be able to acquire tolerance within a generation that persists into later life and subsequent generations, potentially driven by non-genetic mechanisms including epigenetic effects, parental effects, or a combination of these. However, the role of these mechanisms in the response of organisms to intermittent exposure scenarios is not fully understood.
During my PhD, I aimed to investigate whether exposure to common aquatic pollutants would cause altered tolerance to future exposure in later life and in subsequent generations, and explore the potential non-genetic mechanisms responsible. To address this, I used two model fish species – the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) – to examine the molecular and physiological responses of fish following pre-exposure and subsequent re-exposure to a chemical both within and across generations.
First, I investigated the effects of repeated exposure to an environmentally relevant concentration of bisphenol a (BPA) – a ubiquitous endocrine disrupting chemical known to disrupt reproduction and epigenetic regulation – by comparing the response of adult zebrafish and their offspring to BPA following either a single exposure of adults or a pre-exposure followed by a subsequent re-exposure. Although no effects were observed for reproductive output, upon exposure to BPA, transcription of anti-Mullerian hormone (amh) – a hormone involved in oocyte maturation – was found to be significantly downregulated in the ovaries of pre-exposed fish compared to naïve fish experiencing BPA exposure for the first time. In addition, embryonic exposure of offspring demonstrated a protective effect of parental pre-exposure, with offspring of naïve parents being significantly more susceptible to BPA toxicity compared to offspring of pre-exposed parents, which were no more susceptible than offspring of controls. These observations suggest that pre-exposure of adults to BPA led to an altered molecular response upon re-exposure. As well as altering the regulation of amh, I hypothesised that altered physiology of adults likely inferred BPA tolerance to their offspring by modifying the environment in the gonad during gametogenesis.
I then investigated the effects of pre-exposure to copper during embryogenesis on transcriptomic response to re-exposure in later life and on the copper tolerance of subsequent generations. Copper is an essential element that becomes toxic at high concentrations, and is frequently found at toxic levels in freshwater environments. Previous work had shown that pre-exposure of three-spined stickleback to sub lethal copper concentrations during embryogenesis resulted in altered copper storage in tissues in later life despite no further exposure, and caused increased copper uptake in the gill upon re-exposure, indicating lasting physiological changes as a result of historic copper exposure. To investigate this differential physiological response, I conducted RNA-sequencing in the gill of naïve and pre-exposed fish after re-exposure. Both populations exhibited a differential transcriptional response, but this response was more pronounced in naïve fish despite having a lower copper burden in their tissues. The reduced transcriptomic response in pre-exposed fish, suggests that copper caused less disturbance of homeostasis compared to naïve fish. Analysis of differential gene expression suggested that naïve fish experienced greater toxicity upon exposure to copper compared to pre-exposed fish, and may be less able to activate compensatory mechanisms effectively. This included evidence of increased oxidative stress; less effective compensatory transcriptional regulation of ion transporters to prevent ionoregulatory disturbance; and significantly greater cellular proliferation, which may suggest greater incidence of harmful epithelial thickening in the gill. I hypothesised that this differential response may be driven by epigenetic alterations induced by pre-exposure during embryogenesis – which encompasses sensitive windows of epigenetic reprogramming – leading to altered regulation of copper-responsive genes such that they are differentially expressed upon subsequent exposure to copper, inferring increased tolerance.
In addition to an altered response within a generation, previous work also showed that pre-exposure of the F0 generation led to increased copper tolerance in offspring that was maintained to the F2 generation. In order to investigate the longevity of this effect, I exposed F3 embryos from the naive and pre-exposed populations. In contrast to previous generations, F3 embryos were less tolerant to copper exposure compared to controls. This suggests that different mechanisms may be acting in different generations, potentially including both parental effects and transgenerational epigenetic inheritance. I then repeated the F0 and F1 copper exposures in zebrafish, and found that these effects were not conserved, indicating that the mechanisms underpinning acquisition of copper tolerance differ between species.
These data highlight the potential for populations to exhibit differential responses to chemical stressors depending upon exposure history. This has implications for both chemical risk assessment and wildlife management strategies, such as restocking programmes, which should consider the potential for wild populations to become locally adapted via non-genetic mechanisms over short timescales, including via the epigenome. The emergence of latent toxic effects potentially caused by transgenerational epigenetic inheritance also advocates a need to test for potential multigenerational effects in chemical assessment that are not currently routinely investigated.

Aquaculture currently accounts for approximately half of all seafood produced and is the fastest growing farmed food sector globally. Marine bivalve aquaculture, the farming of oysters, mussels and clams, represents a highly sustainable component of this industry and has major potential for global expansion via increased efficiency, and numbers of, production systems. Artificial spat propagation (i.e. settled juveniles) in hatcheries and selective breeding have the potential to offer rapid and widespread gains for molluscan aquaculture industry. However, bivalves have unique life-histories, genetic and genomic characteristics, which present significant challenges to achieving such genetic improvement. Selection pressures experienced by bivalve larvae and spat in the wild contribute to drive population structure and animal fitness. Similarly, domestication selection is likely to act on hatchery-produced spat, the full implications of which have not been fully explored. In this review, we outline the key features of these taxa and production practices applied in bivalve aquaculture, which have the potential to affect the genetic and phenotypic variability of hatchery-propagated stock. Alongside, we compare artificial and natural processes experienced by bivalves to investigate the possible consequences of hatchery propagation on stock production. In addition, we identify key areas of investigation that need to be prioritized to continue to the advancement of bivalve genetic improvement via selective breeding. The growing accessibility of next-generation sequencing technology and high-powered computational capabilities facilitate the implementation of novel genomic tools in breeding programmes of aquatic species. These emerging techniques represent an exciting opportunity for sustainably expanding the bivalve aquaculture sector.

Clozapine is an atypical antipsychotic medication that is used to treat schizophrenia patients who are resistant to other antipsychotic drugs. The molecular mechanisms mediating the effects of clozapine are not well understood and its use is often associated with severe side-effects. In this study, we exposed groups of wild-type zebrafish to two doses of clozapine ('low' (20 µg/L) and 'high' (70 µg/L)) over a 72-h period, observing dose-dependent effects on behaviour. Using RNA sequencing (RNA-seq) we identified multiple genes differentially expressed in the zebrafish brain following exposure to clozapine. Network analysis identified co-expression modules characterised by striking changes in module connectivity in response to clozapine, and these were enriched for regulatory pathways relevant to the etiology of schizophrenia. Our study highlights the utility of zebrafish as a model for assessing the molecular consequences of antipsychotic medications and identifies genomic networks potentially involved in schizophrenia.

Silver is a non-essential, toxic metal widespread in freshwaters and capable of causing adverse effects to wildlife. Its toxic effects have been studied in detail but less is known about how sensitivity varies during development and whether pre-exposures affect tolerance upon re-exposure. We address these knowledge gaps using the zebrafish embryo (Danio rerio) model to investigate whether exposures encompassing stages of development prior to mid-blastula transition, when chorion hardening and epigenetic reprogramming occur, result in greater toxicity compared to those initiated after this period. We conducted exposures to silver initiated at 0.5 h post fertilisation (hpf) and 4 hpf to determine if toxicity differed. In parallel, we exposed embryos to the methylation inhibitor 5-azacytidine as a positive control. Toxicity increased when exposures started from 0.5 hpf compared to 4 hpf and LC50 were significantly lower by 1.2 and 7.6 times for silver and 5-azacyitidine, respectively. We then investigated whether pre-exposure to silver during early development (from 0.5 or 4 hpf) affected the outcome of subsequent exposures during the larvae stage, and found no alterations in toxicity compared to naïve larvae. Together, these data demonstrate that during early development zebrafish embryos are more sensitive to silver when experiments are initiated at the one-cell stage, but that pre-exposures do not influence the outcome of subsequent exposures, suggesting that no long-lasting memory capable of influencing future susceptibility was maintained under our experimental conditions. The finding that toxicity is greater for exposures initiated at the one-cell stage has implications for designing testing systems to assess chemical toxicity.

Aquaculture is the fastest-growing farmed food sector and will soon become the primary source of fish and shellfish for human diets. In contrast to crop and livestock production, aquaculture production is derived from numerous, exceptionally diverse species that are typically in the early stages of domestication. Genetic improvement of production traits via well-designed, managed breeding programmes has great potential to help meet the rising seafood demand driven by human population growth. Supported by continuous advances in sequencing and bioinformatics, genomics is increasingly being applied across the broad range of aquaculture species and at all stages of the domestication process to optimize selective breeding. In the future, combining genomic selection with biotechnological innovations, such as genome editing and surrogate broodstock technologies, may further expedite genetic improvement in aquaculture.

Hypoxia is a major stressor in aquatic environments and it is frequently linked with excess nutrients resulting from sewage effluent discharges and agricultural runoff, which often also contain complex mixtures of chemicals. Despite this, interactions between hypoxia and chemical toxicity are poorly understood. We exposed male three-spined stickleback during the onset of sexual maturation to a model anti-androgen (flutamide; 250 μg/L) and a pesticide with anti-androgenic activity (linuron; 250 μg/L), under either 97% or 56% air saturation (AS). We assessed the effects of each chemical, alone and in combination with reduced oxygen concentration, by measuring the transcription of spiggin in the kidney, as a marker of androgen signalling, and 11 genes in the liver involved in some of the molecular pathways hypothesised to be affected by the exposures. Spiggin transcription was strongly inhibited by flutamide under both AS conditions. In contrast, for linuron, a strong inhibition of spiggin was observed under 97% AS, but this effect was supressed under reduced air saturation, likely due to interactions between the hypoxia inducible factor and the aryl hydrocarbon receptor (AhR) pathways. In the liver, hypoxia inducible factor 1α was induced following exposure to both flutamide and linuron, however this was independent of the level of air saturation. This work illustrates the potential for interactions between hypoxia and pollutants with endocrine or AhR agonist activity to occur, with implications for risk assessment and management.

The aquaculture industry has a substantial role to play in securing future food supply, but its expansion is currently limited by disease. In shrimp aquaculture White Spot Disease (WSD) outbreaks, caused by White Spot Syndrome Virus (WSSV), are the major limiter as they rapidly culminate in 100% mortality and lack prophylactics or therapeutics. Losses associated with WSD are estimated at $1 billion USD annually. All crustaceans are considered to be susceptible to WSSV but the degree of susceptibility varies dramatically between species from the highly susceptible penaeid shrimp to highly resistant European shore crab (Carcinus maenas), which is capable of harbouring the virus for long periods of time without any disease symptoms. This differential susceptibility between crustacean hosts offers the opportunity to investigate how they differ in their molecular responses to WSSV, in an attempt to identify the molecular mechanisms responsible for susceptibility to this pathogen. The thesis therefore aimed to investigate the host-WSSV molecular interactions occurring within two crustaceans with differing susceptibility to WSD in order to construct hypotheses for the likely molecular pathways that could be responsible for tolerance to WSD in crustaceans. Further, the thesis also explored the role of environmental abiotic factors in WSSV susceptibility. To address these aims I first conducted an in-depth literature review of the impacts of the environment on WSD outbreaks. I identified links between rapidly changing environmental conditions and WSD occurrence and substantial gaps in the study of many single and combined factors including dissolved oxygen, CO2, pH and nitrogen concentrations. Secondly, a WSSV infection trial was carried out in (highly susceptible) Penaeusvannamei and (mRNA and miRNA) transcriptomes produced. Transcriptome analysis of susceptible P. vannamei pointed towards increased susceptibility via rapid subversion of processes to enhance WSSV entry, evasion of cellular recycling components and cellular stress with a clear absence of significant immune responses during the early stages of the disease. During early time points, differentially expressed miRNAs were predicted to regulate cytoskeleton polymerisation, phagocytic activity, osmoregulation and metabolism with a hypothesised role in enhancing WSSV infection. The reduced immune responses observed in shrimp may be partially explained by differentially expressed miRNAs such as pPva-miR-H, which reduced immune- priming transcript expression (Dscam) and pPva-miR-K and –N, which regulated apoptosis at 24 and 36 hours post injection (hpi) to favour the virus. The ability of miRNAs to also enhance nucleotide and lipid metabolism from 24 hpi likely further supports virus replication. The P. vannamei transcriptome was then compared to an equivalent dataset in resistant C. maenas, which showed limited transcriptional responses during early time points. Critical alterations to transcripts involved in endocytosis and innate immunity, suggested that crabs may exhibit an enhanced ability to prevent WSSV entry and clear invading virions and that the associated transcripts may be key to resisting WSD. In particular, the significant upregulation of Cma-miR-92b, which may negatively regulate the translation of viral E3 ligase, immediate-early protein in WSSV-recalcitrant crabs should be explored further. For both susceptible and resistant crustaceans many novel miRNAs were identified, contributing to the current lack of crustacean genomic resources. These require further study to determine their significance in the WSSV infection. Based on the comparison of the transcriptome profiles between these two species with contrasting susceptibility to WSD, I propose the hypothesis that a two-pronged mechanism of reduced virion endocytosis (resulting from the inhibition of internal vesicle budding by dynamin-1), downregulation of transcripts that mediate cellular transport and increased viral clearance in crabs not replicating WSSV (by upregulation of apoptosis-associated transcripts) may underpin the mechanism of WSD resistance in crabs. In contrast, I hypothesise that the increased susceptibility of shrimp to WSD arises due to the suppression of both the innate immune responses during early infection and immune-priming molecule Dscam (by pPva-miR-H) during late infection. Further exploration of these key components in the molecular response to WSSV may lead to potential avenues to explore treatments for this devastating disease in aquaculture.

Hypoxia is one of the major threats to biodiversity in aquatic systems. The association of hypoxia with nutrient-rich effluent input into aquatic systems results in scenarios where hypoxic waters could be contaminated with a wide range of chemicals, including metals. Despite this, little is known about the ability of fish to respond to hypoxia when exposures occur in the presence of environmental toxicants. We address this knowledge gap by investigating the effects of exposures to different levels of oxygen in the presence or absence of copper using the three-spined sticklebacks (Gasterosteus aculeatus) model. Fish were exposed to different air saturations (AS; 100%, 75% and 50%) in combination with copper (20 μg/L) over a 4 day period. The critical oxygen level (Pcrit), an indicator of acute hypoxia tolerance, was 54.64 ± 2.51% AS under control conditions, and 36.21 ± 2.14% when fish were chronically exposed to hypoxia (50% AS) for 4 days, revealing the ability of fish to acclimate to low oxygen conditions. Importantly, the additional exposure to copper (20 μg/L) prevented this improvement in Pcrit, impairing hypoxia acclimation. In addition, an increase in ventilation rate was observed for combined copper and hypoxia exposure, compared to the single stressors or the controls. Interestingly, in the groups exposed to copper, a large increase in variation in the measured Pcrit was observed between individuals, both under normoxic and hypoxic conditions. This variation, if observed in wild populations, may lead to selection for a tolerant phenotype and alterations in the gene pool of the populations, with consequences for their sustainability. Our findings provide strong evidence that copper reduces the capacity of fish to respond to hypoxia by preventing acclimation and will inform predictions of the consequences of global increases of hypoxia in water systems affected by other pollutants worldwide.

Populations of fishes provide valuable services for billions of people, but face diverse and interacting threats that jeopardize their sustainability. Human population growth and intensifying resource use for food, water, energy and goods are compromising fish populations through a variety of mechanisms, including overfishing, habitat degradation and declines in water quality. The important challenges raised by these issues have been recognized and have led to considerable advances over past decades in managing and mitigating threats to fishes worldwide. In this review, we identify the major threats faced by fish populations alongside recent advances that are helping to address these issues. There are very significant efforts worldwide directed towards ensuring a sustainable future for the world's fishes and fisheries and those who rely on them. Although considerable challenges remain, by drawing attention to successful mitigation of threats to fish and fisheries we hope to provide the encouragement and direction that will allow these challenges to be overcome in the future.

Survival of marine fishes that are exposed to elevated near-future CO2 levels is threatened by their altered responses to sensory cues. Here we demonstrate a physiological and molecular mechanism in the olfactory system that helps to explain altered behaviour under elevated CO2. We combine electrophysiology measurements and transcriptomics with behavioural experiments to investigate how elevated CO2 affects the olfactory system of European sea bass (Dicentrarchus labrax). When exposed to elevated CO2 (approximately 1,000 µatm), fish must be up to 42% closer to an odour source for detection, compared with current CO2 levels (around 400 µatm), decreasing their chances of detecting food or predators. Compromised olfaction correlated with the suppression of the transcription of genes involved in synaptic strength, cell excitability and wiring of the olfactory system in response to sustained exposure to elevated CO2 levels. Our findings complement the previously proposed impairment of γ-aminobutyric acid receptors, and indicate that both the olfactory system and central brain function are compromised by elevated CO2 levels.

Reproduction is an essential process for life and is regulated by complex hormone networks and environmental factors. To date, little is known about the contribution of epigenetic mechanisms to the regulation of reproduction, particularly in lower vertebrates. We used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. We found evidence for associations between DNA promotor methylation and transcription for esr1 (gonads and female livers), amh (gonads) and dnmt1 (livers). In the liver, esr1 was shown to be significantly over-expressed in females compared to males, and its promoter was significantly hypo-methylated in females compared to males. In the gonads, genes involved in epigenetic processes including dnmt1, dnmt3 and hdac1 were over-expressed in the ovary compared to the testis. In addition, dnmt1 and dnmt3 transcription in the testis was found to be strongly correlated with global DNA methylation. These data provide evidence of the sex-specific epigenetic regulation and transcription of genes involved in reproduction and epigenetic signalling in a commonly used vertebrate model.

Over the last decade, advances in high-throughput sequencing technologies have revolutionised biological research, making it possible for DNA/RNA sequencing of any organism of interest to be undertaken. Sequencing approaches are now routinely used in the detection and characterisation of (novel) viruses, investigation of host-pathogen interactions, and effective development of disease treatment strategies. For the sequencing and identification of viruses of interest, metagenomics approaches using infected host tissue are frequently used, as it is not always possible to culture and isolate these pathogens. High-throughput sequencing can also be used to investigate host-pathogen interactions by investigating (temporal) transcriptomic responses of both the host and virus, potentially leading to the discovery of novel opportunities for treatment and drug targets. In addition, viruses in environmental samples (e.g. water or soil samples) can be identified using eDNA/metagenomics approaches. The promise that recent developments in sequencing brings to the field of invertebrate virology are not devoid of technical challenges, including the need for better laboratory and bioinformatics strategies to sequence and assemble virus genomes within complex tissue or environmental samples, and the difficulties associated with the annotation of the large number of novel viruses being discovered.

Diclofenac is one of the most widely prescribed nonsteroidal anti-inflammatory drugs worldwide. It is frequently detected in surface waters; however, whether this pharmaceutical poses a risk to aquatic organisms is debated. Here we quantified the uptake of diclofenac by the fathead minnow (Pimephales promelas) following aqueous exposure (0.2-25.0 μg L-1) for 21 days, and evaluated the tissue and biomolecular responses in the kidney. Diclofenac accumulated in a concentration- and time-dependent manner in the plasma of exposed fish. The highest plasma concentration observed (for fish exposed to 25 μg L-1 diclofenac) was within the therapeutic range for humans. There was a strong positive correlation between exposure concentration and the number of developing nephrons observed in the posterior kidney. Diclofenac was not found to modulate the expression of genes in the kidney associated with its primary mode of action in mammals (prostaglandin-endoperoxide synthases) but modulated genes associated with kidney repair and regeneration. There were no significant adverse effects following 21 days exposure to concentrations typical of surface waters. The combination of diclofenac's uptake potential, effects on kidney nephrons and relatively small safety margin for some surface waters may warrant a longer term chronic health effects analysis for diclofenac in fish.

Hypoxia is a global problem in aquatic systems and often co-occurs with pollutants. Despite this, little is known about the combined effects of these stressors on aquatic organisms. The objective of this study was to investigate the combined effects of hypoxia and copper, a toxic metal widespread in the aquatic environment. We used the three-spined stickleback (Gasterosteus aculeatus) as a model because of its environmental relevance and amenability for environmental toxicology studies. We focused on embryonic development as this is considered to be a sensitive life stage to environmental pollution. We first investigated the effects of hypoxia alone on stickleback development to generate the information required to design subsequent studies. Our data showed that exposure to low oxygen concentrations (24.7 ± 0.9% air saturation; AS) resulted in strong developmental delays and increased mortalities, whereas a small decrease in oxygen (75.0 ± 0.5%AS) resulted in premature hatching. Stickleback embryos were then exposed to a range of copper concentrations under hypoxia (56.1 ± 0.2%AS) or normoxia (97.6 ± 0.1%AS), continuously, from fertilisation to free swimming larvae. Hypoxia caused significant changes in copper toxicity throughout embryonic development. Prior to hatching, hypoxia suppressed the occurrence of mortalities, but after hatching hypoxia significantly increased copper toxicity. Interestingly, when exposures were conducted only after hatching, the onset of copper-induced mortalities was delayed under hypoxia compared to normoxia, but after 48 h, copper was more toxic to hatched embryos under hypoxia. This is the second species for which the protective effect of hypoxia on copper toxicity prior to hatching, followed by its exacerbating effect after hatching is demonstrated, suggesting the hypothesis that this pattern may be common for teleost species. Our research highlights the importance of considering the interactions between multiple stressors, as understanding these interactions is essential to facilitate the accurate prediction of the consequences of exposure to complex stressors in a rapidly changing environment.

Some fish populations inhabiting contaminated environments show evidence of increased chemical tolerance, however the mechanisms contributing to this tolerance, and whether this is heritable, are poorly understood. We investigated the responses of two populations of wild three-spined stickleback (Gasterosteus aculeatus) with different histories of contaminant exposure to an oestrogen and copper, two widespread aquatic pollutants. Male stickleback originating from two sites, the River Aire, with a history of complex pollution discharges, and Siblyback Lake, with a history of metal contamination, were depurated and then exposed to copper (46μg/L) and the synthetic oestrogen ethinyloestradiol (22ng/L). The hepatic transcriptomic response was compared between the two populations and to a reference population with no known history of exposure (Houghton Springs, Dorset). Gene responses included those typical for both copper and oestrogen, with no discernable difference in response to oestrogen between populations. There was, however, some difference in the magnitude of response to copper between populations. Siblyback fish showed an elevated baseline transcription of genes encoding metallothioneins and a lower level of metallothionein induction following copper exposure, compared to those from the River Aire. Similarly, a further experiment with an F1 generation of Siblyback fish bred in the laboratory found evidence for elevated transcription of genes encoding metallothioneins in unexposed fish, together with an altered transcriptional response to 125μg/L copper, compared with F1 fish originating from the clean reference population exposed to the same copper concentration. These data suggest that the stickleback from Siblyback Lake have a differential response to copper, which is inherited by the F1 generation in laboratory conditions, and for which the underlying mechanism may include an elevation of baseline transcription of genes encoding metallothioneins. The genetic and/or epigenetic mechanisms contributing to this inherited alteration of metallothionein transcription have yet to be established.

Bisphenol a (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment.

Hypoxia is a global and increasingly important stressor in aquatic ecosystems, with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a wide range of chemicals but little is known about the interactions between these stressors. We investigated the effects of hypoxia on the responses of zebrafish (Danio rerio) embryos to copper, a widespread aquatic contaminant. We showed that during continuous exposures copper toxicity was reduced by over 2-fold under hypoxia compared to normoxia. When exposures were conducted during 24 h windows, hypoxia reduced copper toxicity during early development and increased its toxicity in hatched larvae. To investigate the role of the hypoxia signaling pathway on the suppression of copper toxicity during early development, we stabilized the hypoxia inducible factor (HIF) pathway under normoxia using a prolyl-4-hydroxylase inhibitor, dimethyloxalylglycine (DMOG) and demonstrated that HIF activation results in a strong reduction in copper toxicity. We also established that the reduction in copper toxicity during early development was independent of copper uptake, while after hatching, copper uptake was increased under hypoxia, corresponding to an increase in copper toxicity. These findings change our understanding of the current and future impacts of worldwide oxygen depletion on fish communities challenged by anthropogenic toxicants.

Since its emergence in the 1990s, White Spot Disease (WSD) has had major economic and societal impact in the crustacean aquaculture sector. Over the years shrimp farming alone has experienced billion dollar losses through WSD. The disease is caused by the White Spot Syndrome Virus (WSSV), a large dsDNA virus and the only member of the Nimaviridae family. Susceptibility to WSSV in a wide range of crustacean hosts makes it a major risk factor in the translocation of live animals and in commodity products. Currently there are no effective treatments for this disease. Understanding the molecular basis of disease processes has contributed significantly to the treatment of many human and animal pathogens, and with a similar aim considerable efforts have been directed towards understanding host-pathogen molecular interactions for WSD. Work on the molecular mechanisms of pathogenesis in aquatic crustaceans has been restricted by a lack of sequenced and annotated genomes for host species. Nevertheless, some of the key host-pathogen interactions have been established: between viral envelope proteins and host cell receptors at initiation of infection, involvement of various immune system pathways in response to WSSV, and the roles of various host and virus miRNAs in mitigation or progression of disease. Despite these advances, many fundamental knowledge gaps remain; for example, the roles of the majority of WSSV proteins are still unknown. In this review we assess current knowledge of how WSSV infects and replicates in its host, and critique strategies for WSD treatment.

BACKGROUND: the European shore crab, Carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. It is also an important invasive species in numerous global locations. However, the genomic resources for this organism are still sparse, limiting research progress in these fields. To address this resource shortfall we produced a C. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species. RESULTS: We isolated and pooled RNA for twelve different tissues and organs from C. maenas individuals and sequenced the RNA using next generation sequencing on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome was generated encompassing 212,427 transcripts (153,699 loci). The transcripts were filtered, annotated and characterised using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including NCBI, Gene Ontology and KEGG). There were differential patterns of expression for between 1,223 and 2,741 transcripts across tissues and organs with over-represented Gene Ontology terms relating to their specific function. Based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the RNAi, Toll-like receptor signalling, IMD and JAK/STAT pathways. CONCLUSIONS: We have produced an assembled transcriptome for C. maenas that provides a significant molecular resource for wide ranging studies in this species. Analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns.

BACKGROUND: Glyphosate, the active ingredient in Roundup formulations, is the most widely used herbicide worldwide, and as a result contaminates surface waters and has been detected in food residues, drinking water and human urine, raising concerns for potential environmental and human health impacts. Research has shown that glyphosate and Roundup can induce a broad range of biological effects in exposed organisms, particularly via generation of oxidative stress. However, there has been no comprehensive investigation of the global molecular mechanisms of toxicity of glyphosate and Roundup for any species. We aimed to characterise and compare the global mechanisms of toxicity of glyphosate and Roundup in the liver of brown trout (Salmo trutta), an ecologically and economically important vertebrate species, using RNA-seq on an Illumina HiSeq 2500 platform. To do this, we exposed juvenile female brown trout to 0, 0.01, 0.5 and 10 mg/L of glyphosate and Roundup (glyphosate acid equivalent) for 14 days, and sequenced 6 replicate liver samples from each treatment. RESULTS: We assembled the brown trout transcriptome using an optimised de novo approach, and subsequent differential expression analysis identified a total of 1020 differentially-regulated transcripts across all treatments. These included transcripts encoding components of the antioxidant system, a number of stress-response proteins and pro-apoptotic signalling molecules. Functional analysis also revealed over-representation of pathways involved in regulating of cell-proliferation and turnover, and up-regulation of energy metabolism and other metabolic processes. CONCLUSIONS: These transcriptional changes are consistent with generation of oxidative stress and the widespread induction of compensatory cellular stress response pathways. The mechanisms of toxicity identified were similar across both glyphosate and Roundup treatments, including for environmentally relevant concentrations. The significant alterations in transcript expression observed at the lowest concentrations tested raises concerns for the potential toxicity of this herbicide to fish populations inhabiting contaminated rivers.

Estrogenic chemicals are major contaminants of surface waters and can threaten the sustainability of natural fish populations. Characterization of the global molecular mechanisms of toxicity of environmental contaminants has been conducted primarily in model species rather than species with limited existing transcriptomic or genomic sequence information. We aimed to investigate the global mechanisms of toxicity of an endocrine disrupting chemical of environmental concern [17β-estradiol (E2)] using high-throughput RNA sequencing (RNA-Seq) in an environmentally relevant species, brown trout (Salmo trutta). We exposed mature males to measured concentrations of 1.94, 18.06, and 34.38 ng E2/l for 4 days and sequenced three individual liver samples per treatment using an Illumina HiSeq 2500 platform. Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05). Functional analysis revealed upregulation of processes associated with vitellogenesis, including lipid metabolism, cellular proliferation, and ribosome biogenesis, together with a downregulation of carbohydrate metabolism. Using real-time quantitative PCR, we validated the expression of eight target genes and identified significant differences in the regulation of several known estrogen-responsive transcripts in fish exposed to the lower treatment concentrations (including esr1 and zp2.5). We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l. These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available.

The herbicide linuron is used worldwide, and has been detected in surface waters as well as in food and drinking water. Toxicological studies have reported that linuron acts as an antiandrogen in vitro and in vivo and disrupts mammalian male reproductive function. However, global mechanisms of linuron toxicity are poorly documented. We used RNA-seq to characterize the hepatic transcriptional response of mature male brown trout exposed for 4 days to 1.7, 15.3, and 225.9 μg/L linuron. We identified a striking decrease in the expression of transcripts encoding the majority of enzymes forming the cholesterol biosynthesis pathway. We also measured a very significant decrease in total hepatic cholesterol in fish exposed to 225.9 μg/L linuron and a negative correlation between total cholesterol and linuron treatment concentration. We hypothesize that inhibition of cholesterol biosynthesis may result from the disruption of androgen signaling by linuron. Additionally, there was increased expression of a number of transcripts involved in cellular stress responses, including cyp1a (up to 560-fold), molecular chaperones, and antioxidant enzymes. We found some evidence of similar patterns of transcriptional change in fish exposed to an environmentally relevant concentration of linuron, and further research should investigate the potential for adverse effects to occur following chronic environmental exposure.

Roundup and its active ingredient glyphosate are among the most widely used herbicides worldwide and may contaminate surface waters. Research suggests both Roundup and glyphosate induce oxidative stress in fish and may also cause reproductive toxicity in mammalian systems. We aimed to investigate the reproductive effects of Roundup and glyphosate in fish and the potential associated mechanisms of toxicity. To do this, we conducted a 21-day exposure of breeding zebrafish (Danio rerio) to 0.01, 0.5, and 10 mg/L (glyphosate acid equivalent) Roundup and 10 mg/L glyphosate. 10 mg/L glyphosate reduced egg production but not fertilization rate in breeding colonies. Both 10 mg/L Roundup and glyphosate increased early stage embryo mortalities and premature hatching. However, exposure during embryogenesis alone did not increase embryo mortality, suggesting that this effect was caused primarily by exposure during gametogenesis. Transcript profiling of the gonads revealed 10 mg/L Roundup and glyphosate induced changes in the expression of cyp19a1 and esr1 in the ovary and hsd3b2, cat, and sod1 in the testis. Our results demonstrate that these chemicals cause reproductive toxicity in zebrafish, although only at high concentrations unlikely to occur in the environment, and likely mechanisms of toxicity include disruption of the steroidogenic biosynthesis pathway and oxidative stress.

Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis with whole-transcriptome profiling using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively unimpacted river, were significantly increased in the gills, liver and kidney (63-, 34- and 19-fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.

Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity, however, remain unclear. We examined the effects of exposure to silver in nano-, bulk-, and ionic forms on zebrafish embryos (Danio rerio) using a Next Generation Sequencing approach in an Illumina platform (High-Throughput SuperSAGE). Significant alterations in gene expression were found for all treatments and many of the gene pathways affected, most notably those associated with oxidative phosphorylation and protein synthesis, overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down-regulation of this pathway at 24 h of exposure, but with a recovery at 48 h. This finding was consistent with a dose-dependent decrease in oxygen consumption at 24 h, but not at 48 h, following exposure to silver ions. Overall, our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms.

Pollution is a significant environmental pressure on fish populations in both freshwater and marine environments. Populations subjected to chronic exposure to pollutants can experience impacts ranging from altered reproductive capacity to changes in population genetic structure. Few studies, however, have examined the reproductive vigor of individuals within populations inhabiting environments characterized by chronic pollution. In this study we undertook an analysis of populations of three-spined sticklebacks (Gasterosteus aculeatus) from polluted sites, to determine levels of genetic diversity, assess for evidence of historic population genetic bottlenecks and determine the reproductive competitiveness of males from these locations. The sites chosen included locations in the River Aire, the River Tees and the River Birket, English rivers that have been impacted by pollution from industrial and/or domestic effluents for over 100 years. Male reproductive competitiveness was determined via competitive breeding experiments with males and females derived from a clean water site, employing DNA microsatellites to determine parentage outcome. Populations of stickleback collected from the three historically polluted sites showed evidence of recent population bottlenecks, although only the River Aire population showed low genetic diversity. In contrast, fish collected from two relatively unpolluted sites within the River Gowy and Houghton Springs showed weak, or no evidence of such bottlenecks. Nevertheless, males derived from polluted sites were able to reproduce successfully in competition with males derived from clean water exposures, indicating that these bottlenecks have not resulted in any substantial loss of reproductive fitness in males. © 2013 Elsevier B.V.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is frequently present in indoor dust and can be detected in human milk. In order to evaluate the effects of TDCPP on vertebrate development, zebrafish embryos/larvae were used as an animal model to examine developmental phenotypes and explore possible mechanisms of toxicity by employing microarrays and iTRAQ labeling quantitative proteomics. The results demonstrated that treatment with TDCPP (3 μM) from 0.75 h postfertilization (hpf) inhibited cell rearrangement at 4 hpf, caused delay in epiboly at 5.7 and 8.5 hpf, and led to abnormal development (e.g. short tail, reduced body size) and lethality between 14 and 45 hpf, which might be related with altered expression of genes regulating embryogenesis. Furthermore, trunk curvature was observed as the main phenotype in 96 hpf zebrafish larvae exposed to 1 or 3 μM TDCPP, possibly by changing somite formation and expression of proteins related to fast muscle and cartilage development. Collectively, our results suggest that exposure to TDCPP causes developmental toxicity in vertebrates and warrant the need for studies to evaluate the potential health risks of TDCPP to developing human embryos/infants/children, due to its frequent presence in indoor dust and potential for human exposure.

The intestine of marine teleosts produces carbonate precipitates from ingested calcium as part of their osmoregulatory strategy in seawater. The potential for estrogens to control the production of intestinal calcium carbonate and so influence osmoregulation was investigated in seawater-acclimated rainbow trout following intraperitoneal implantation of 17β-estradiol (E2) at two doses (0.1 and 10 μg E2 g(-1)). Levels of plasma vitellogenin provided an indicator of estrogenic effect, increasing significantly by three and four orders of magnitude at the low and high doses, respectively. Plasma osmolality and muscle water content were unaffected, whereas E2-treated fish maintained lower plasma [Na(+)] and [Cl(-)]. Plasma [Ca(2+)] and [Mg(2+)] and muscle [Ca(2+)] increased with vitellogenin induction, whereas the intestinal excretion of calcium carbonate was reduced. This suggests that elevated levels of circulating E2 may enhance Ca(2+) uptake via the gut and simultaneously reduce CaCO(3) formation, which normally limits intestinal availability of Ca(2+). Increasing E2 caused an elevation of [Na(+)] and [Cl(-)] and a reduction of [HCO(3(-))] in intestinal fluid. We speculate that E2 may influence a number of intestinal ion transport processes that ultimately may influence water absorption: (1) reduced NaCl cotransport, (2) reduced Cl(-) uptake via Cl(-)/HCO(3(-)) exchange and (3) reduced precipitation of Ca(2+) and Mg(2+) carbonates. Despite these effects on intestinal ion and water transport, overall osmoregulatory status was not compromised in E2-treated fish, suggesting the possibility of compensation by other organs.

Despite increasing application of silver nanoparticles (NPs) in industry and consumer products, there is still little known about their potential toxicity, particularly to organisms in aquatic environments. To investigate the fate and effects of silver NPs in fish, rainbow trout (Oncorhynchus mykiss) were exposed via the water to commercial silver particles of three nominal sizes: 10 nm (N(10)), 35 nm (N(35)), and 600-1600 nm (N(Bulk)), and to silver nitrate for 10 days. Uptake into the gills, liver, and kidneys was quantified by inductively coupled plasma-optical emission spectrometry, and levels of lipid peroxidation in gills, liver, and blood were determined by measurements of thiobarbituric acid reactive substances. Expression of a suite of genes, namely cyp1a2, cyp3a45, hsp70a, gpx, and g6pd, known to be involved in a range of toxicological response to xenobiotics was analyzed in the gills and liver using real-time PCR. Uptake of silver particles from the water into the tissues of exposed fish was low but nevertheless occurred for current estimated environmental exposures. of the silver particles tested, N(10) were found to be the most highly concentrated within gill tissues and N(10) and N(Bulk) were the most highly concentrated in liver. There were no effects on lipid peroxidation in any of the tissues analyzed for any of the silver particles tested, and this is likely due to the low uptake rates. However, exposure to N(10) particles was found to induce expression of cyp1a2 in the gills, suggesting a possible increase in oxidative metabolism in this tissue.

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.

Copper (Cu) is a micronutrient essential for the biochemical functioning of numerous processes in vertebrates but is also often present in the aquatic environment at concentrations able to cause adverse health effects in aquatic organisms. This study investigated the signaling pathways mediating the effects of exposure to Cu using a toxicogenomic approach in a fish model, the stickleback ( Gasterosteus aculeatus ). Freshwater-acclimated male fish were exposed via the water to Cu, including at environmentally relevant concentrations (3.2-128 microg of Cu/L for 4 days), and the biological responses explored through analyses of the hepatic transcriptome and metabolome and phenotypic end points, including assessment of DNA damage in blood cells. The Cu exposures resulted in DNA strand breaks in blood cells at all exposure concentrations and alterations in hepatic gene expression and metabolite concentrations in a concentration-dependent manner (from 10 microg of Cu/L). Genes associated with the cholesterol biosynthesis pathway were significantly over-represented and consistently down-regulated (at 128 microg of Cu/L), similar to that occurring in a mouse model for Wilson's disease. Additionally, inductions in metallothionein and catalase were also observed. The concentrations of NAD(+) and lactate increased significantly with the Cu exposure, consistent with a shift toward anaerobic metabolism, and these aligned closely with changes observed in gene expression. The pathways of Cu toxicity identified in our study support the conserved mechanisms of Cu toxicity from lower vertebrates to mammals, provide novel insights into the deleterious effects of Cu in fish, and further demonstrate the utility of fish as environmental sentinels for chemical impacts on both environmental and human health.

BACKGROUND: in this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: the consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: the participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Phthalates are ubiquitous in the aquatic environment and are known to adversely affect male reproductive health in mammals through interactions with multiple receptor systems. However, little is known about the risks they pose to fish. This project investigated the effects of di(2-ethylhexyl) phthalate (DEHP), the most commonly used phthalate, on the reproductive health of male zebrafish (Danio rerio). Males were treated with 0.5, 50 and 5000 mg DEHP kg(-1) (body weight) for a period of 10 days via intraperitoneal injection. The effects of the exposure were assessed by analysing fertilisation success, testis histology, sperm DNA integrity and transcript profiles of the liver and testis. A significant increase in the hepatosomatic index and levels of hepatic vitellogenin transcript were observed following exposure to 5000 mg DEHP kg(-1). Exposure to 5000 mg DEHP kg(-1) also resulted in a reduction in fertilisation success of oocytes spawned by untreated females. However, survival and development of the resulting embryos were unaffected by all treatments, and no evidence of DEHP-induced sperm DNA damage was observed. Exposure to 50 and 5000 mg DEHP kg(-1) caused alterations in the proportion of germ cells at specific stages of spermatogenesis in the testis, including a reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes, suggesting that DEHP may inhibit the progression of meiosis. In parallel, exposure to 5000 mg DEHP kg(-1) increased the levels of two peroxisome proliferator-activated receptor (PPAR) responsive genes (acyl-coenzyme a oxidase 1 (acox1) and enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme a dehydrogenase (ehhadh). These data demonstrated that exposure to high concentrations of DEHP disrupts spermatogenesis in adult zebrafish with a consequent decrease in their ability to fertilise oocytes spawned by untreated females. Furthermore, our data suggest that the adverse effects caused by exposure to DEHP are likely to occur preferentially via PPAR signalling pathways in the testis and oestrogen signalling pathways in the liver. We found no evidence of adverse effects on zebrafish reproductive health following exposure to the concentrations occurring in most aquatic systems, indicating that DEHP alone may not be a causative agent of the reproductive abnormalities seen in wildlife, at least as a result of short-term exposures.

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol a (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng 1 -1 to μg 1-1 range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.

A three-spined stickleback (Gasterosteus aculeatus) cDNA array and one-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach, together with individual biomarkers,were employed to investigate the responses of male sticklebacks to polycyclic aromatic hydrocarbon exposure. Fish were exposed to 1,2:5,6-dibenzanthracene (DbA) at concentrations between 0.01 and 50 microg per liter dissolved in the ambient water for four days, and hepatic transcript and metabolite profiles were determined in comparison with those of solvent-exposed controls. Induction of gene expression was apparent for cytochrome P450 1A (CYP1A) and CYP2-family monooxygenases and these responses were strongly correlated with DbA exposure concentrations (for CYP1A r > 0.996). Expression of suites of genes related to bile acid biosynthesis, steroid metabolism, and endocrine function were also affected, as demonstrated by gene ontology analyses. Expression changes in selected genes were confirmed by real-time PCR. Metabolomics highlighted notable changes in concentrations of taurine, malonate, glutamate, and alanine. These statistically significant responses to environmentally relevant concentrations of DbA at the transcriptomic and metabolomic levels provided sensitive markers characteristic of environmentally relevant low-level DbA exposure. Metabolic pathways were identified where both gene expression and metabolite concentrations were altered in response to DbA.

The molecular signalling pathways mediating sexual dimorphism have principally been investigated in the gonads, and to a lesser extent in other organs. The brain plays a central role in coordinating sexual function, including the regulation of reproductive development, maturation and sexual behaviour in both sexes. In this study, we investigated sex-related differences in gene expression in the brains of breeding zebrafish (Danio rerio) to establish a greater understanding of the sex-specific physiology of the brain in lower vertebrates. The brain transcriptomic profiles of males and females were interrogated to identify the genes showing sexually dimorphic gene expression. 42 genes were differentially expressed between the sexes, from which 18 genes were over-expressed in males and 24 genes were over-expressed in females. In males, these included deiodinase, iodothyronine, type II and ribosomal protein S8, and in females, superoxide dismutase [Cu-Zn], sprouty-4, frizzled 10 and testis enhanced gene transcript. Estrogen responsive elements were found in the regulatory regions for 3 genes over-expressed in males and 7 genes over-expressed in females. We have demonstrated the existence of dimorphic patterns of gene expression in the brain of a sexually mature, non-mammalian, vertebrate model, with implications for studies into reproduction and chemical disruption of brain function.

Laboratory tests that quantify reproductive success using model fish species are used to investigate for population-level effects of endocrine disrupting chemicals (EDCs) and other chemicals discharged into the environment. Even for the zebrafish (Danio rerio), however, one of the most widely used laboratory models, surprisingly little is known about the normal variability in measures of reproductive success and this information is crucial for robust test design. In this study, the dynamics of breeding and inherent variability in egg output/viability and sperm quality were characterized among individuals/colonies and over time in 34 colonies of laboratory-kept zebrafish over a 20-day study period. For this work, a '6 x 6' (six males and six females) colony size was adopted, as this is both environmentally relevant and optimal when considering egg output and animal welfare combined: an initial experiment showed egg output per female increased with decreasing colony size however, there was also a parallel increase in aggressive behavior. Both egg output and viability in '6 x 6' colonies were highly variable among colonies (with co-efficients of variation (CVs) of 30 and 11%, respectively) and over the 20-day study duration (considering egg output and viability of all the colonies combined, the CVs were 20 and 12%, respectively). The patterns of egg production also differed among the '6 x 6' colonies, and they included a cyclical output, a consistent daily output, an infrequent egg output with intermittent days of very high egg output, and an output with no obvious pattern. Sperm quality, measured as percentage motility and curvilinear velocity (VCL), was variable both among individuals within '6 x 6' colonies and across colonies, with percentage motility being the most variable parameter (mean CVs of 82% inter-individual within colonies and 49% inter-colony). Sperm quality did not, however, vary over a 24h period. A minimum number of six replicate '6 x 6' colonies, assessed daily for a period of 4 days, was required per treatment to detect a 40% change in egg output. The minimum numbers of individual males required per treatment to detect a 40% change in sperm quality using the breeding system adopted were 32 males for percentage motility and 12 males for VCL, equivalent to six and two '6 x 6' colonies, respectively. These data demonstrate the need for high levels of replication when testing for effects of EDCs on reproductive output in the zebrafish model in an environmentally relevant ('6 x 6') breeding matrix.

Gene expression profiling offers considerable potential for identifying chemical causation of effects induced in exposures to complex mixtures, and for understanding the mechanistic basis for their phenotypic effects. We characterized gene expression responses in livers and gonads of fathead minnow (Pimephales promelas) exposed (for 14-21 days) to estrogenic wastewater treatment works final effluents with varying potencies and assessed the extent to which these expression profiles mapped with those induced by individual steroid estrogens present in the effluents (17beta-estradiol and 17alpha-ethinylestradiol) and, thus, were diagnostic of estrogen exposure. For these studies, we adopted a targeted approach (via real-time PCR) with a suite of 12 genes in liver and 21 genes in gonad known to play key roles in reproduction, growth and development (processes controlled by estrogens) and responses were compared with effects on phenotypic end points indicative of feminization. Gene responses to effluent were induced predominantly in a linear (monotonic) concentration-dependent manner but were complex with many genes responding differently between tissue types and sexes. The gene expression profiles for the estrogenic effluents and the individual steroid estrogens had many common features. There were marked differences in the profiles between the two effluents, however, that were not explained by differences in their estrogenic potencies, suggesting that these may have arisen as a consequence of differences in the contents of other chemicals, which may act directly or indirectly with the estrogen-response pathway to alter estrogen-induced gene expression. These data demonstrate that the patterns of gene expression induced by estrogenic effluents, although complex, can be diagnostic for some of the estrogens they contain and provide insights into the mechanistic basis for the phenotypic effects seen.

Environmental oestrogens are widespread in the aquatic environment and cause alterations in sexual development and function in vertebrates. The molecular pathways underpinning these effects, however, remain poorly understood. In this study, we aimed at generating a mechanistic understanding of the disruptive effects of exposure to environmentally relevant concentrations of 17 alpha-ethinyloestradiol (EE(2)) on reproduction in zebrafish, by anchoring the transcriptomic alterations induced with the physiological consequences of exposure. Breeding colonies of zebrafish were exposed for a 21-day period to three concentrations of EE(2) (0.05, 0.5 and 5 ng/L) and the gonadal transcriptomic alterations induced (determined using a 17,000 oligonucleotide microarray) were analysed together with physiological effects seen on reproductive output of both males and females. Exposure to 5 ng EE(2)/L resulted in reproductive impairment characterised by a decrease in egg production, alterations in sperm quality and reduced fertilisation success. The effects seen were associated with altered expression of 114 and 131 genes in the gonads of males and females, respectively. The biological processes most affected by the exposure were protein metabolism in males and mitochondria organisation and biogenesis in females. Genes involved in the regulation of cell cycle progression, the ubiquitin system and glutathione peroxidase were affected by the EE(2) exposure and associated with the changes observed in gamete quality in both genders. In summary, we demonstrated that EE(2) exposure compromised the reproductive health of breeding zebrafish at environmentally relevant concentrations. The molecular mechanisms mediating some of these effects were identified and included those impacting processes central to gametogenesis in both males and females.

The zebrafish (Danio rerio) is used extensively as a model species for studies on vertebrate development and for assessing chemical effects on reproduction. Despite this, the molecular mechanisms controlling zebrafish reproduction are poorly understood. We analyzed the transcriptomic profiles of the gonads of individual zebrafish, using a 17k oligonucleotide microarray, to define the molecular basis of sex and reproductive status in sexually mature fish. The gonadal transcriptome differed substantially between sexes. Among the genes overexpressed in females, 11 biological processes were overrepresented including mitochondrion organization and biogenesis, and cell growth and/or maintenance. Among the genes overexpressed in males, six biological processes were overrepresented including protein biosynthesis and protein metabolism. Analysis of the expression of gene families known to be involved in reproduction identified a number of genes differentially expressed between ovaries and testes including a number of sox genes and genes belonging to the insulin-like growth factor and the activin-inhibin pathways. Real-time quantitative PCR confirmed the expression profiles for nine of the most differentially expressed genes and indicated that many transcripts are likely to be switched off in one of the sexes in the gonads of adult fish. Significant differences were seen between the gonad transcriptomes of individual reproductively active females reflecting their stage of maturation, whereas the testis transcriptomes were remarkably similar between individuals. In summary, we have identified molecular processes associated with (gonadal) sex specificity in breeding zebrafish and established a strong relationship between individual ovarian transcriptomes and reproductive status in females.

Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW a and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.

Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly established; however, the function(s) of FSH is less certain. The lack of specific and sensitive assays to quantify FSH and its alpha and beta subunits has hindered studies to assess physiological function. In this study, gel filtration chromatography, ion exchange chromatography, and HPLC were employed to purify FSH and its subunits from pituitary glands of rainbow trout (Oncorhynchus mykiss), and the identities of the isolates were confirmed by amino acid analysis. Polyclonal antibodies were raised against the free GTHalpha2 and free FSHbeta subunits to develop specific RIAs. The sensitivities of the intact FSH, GTHalpha2, and FSHbeta assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with each other and with intact LH in the heterologous assays was 90% throughout. Measurement of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTHalpha2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 +/- 6.3 ng/ml in plasma; 37.63 +/- 5.79 microg/pituitary). In both the plasma and the pituitary, free FSHbeta subunit was present throughout the reproductive cycle but at very low concentrations when compared with both free GTHalpha2 and intact FSH. The presence of free GTHalpha2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established.

Female rainbow trout (Oncorhynchus mykiss) were exposed to 4-nonylphenol (NP) at (mean measured) concentrations of 0.7, 8.3, and 85.6 micrograms/L for 18 weeks, during early ovarian development. Fish were sampled sublethally every six weeks, and terminal samples were taken at 18 weeks. NP induced an estrogenic effect (the synthesis of vitellogenin) at concentrations of 8.3 and 85.6 micrograms/L. An effect on gonadotropin synthesis and secretion was also observed. Plasma follicle stimulating hormone (FSH) levels and FSH gene expression in the pituitary were the most sensitive endpoints assessed, being reduced at the lowest dose employed (0.7 microgram NP/L). Pituitary gland luteinizing hormone (LH) content was significantly lower in fish exposed to 85.6 micrograms NP/L, and LH gene expression was suppressed in fish exposed to 8.3 and 85.6 micrograms NP/L. In contrast, plasma LH concentration increased in these fish, but by a very minor absolute amount, and returned to control levels by the final sampling time. Gonadal development ceased in the fish exposed to 85.6 micrograms NP/L, and steroidogenesis in these fish was also markedly inhibited. Although the mechanisms underlying these responses are unknown, this study demonstrates that NP has adverse effects on pituitary function that can result in inhibition of ovarian development.

Female rainbow trout (Oncorhynchus mykiss) produce a single batch of eggs each year; synchronous growth of oocytes, all of which are ovulated at the same time, occurs in the two ovaries. To examine the regulatory mechanisms controlling egg size and number, virgin female rainbow trout were subjected to unilateral ovariectomy (ULO) during early vitellogenesis, and oocyte recruitment and growth in the remaining ovary were monitored. The study also set out to determine whether the presence of a second population of smaller oocytes in the maturing pool (induced by ULO) affected the timing of ovulation and/or the size of the eggs ovulated. Two months after ULO, there was no difference in the gonadosomatic index between ULO fish and controls. Compensatory ovarian hypertrophy resulted from the recruitment of a second population of primary oocytes into the vitellogenic pool. This population of smaller maturing oocytes in the ULO fish displayed growth rates up to twice those of the population of larger oocytes in the same ovary and of oocytes in controls. The growth rate of the population of larger oocytes in the ULO fish was not altered by the recruitment of a second maturing population. One month after ULO, fish had a lower concentration of plasma estradiol-17 beta than did controls; subsequently the concentrations of plasma estradiol-17 beta in the ULO and control groups were similar. After ULO, plasma levels of vitellogenin in the ULO fish did not differ from those in the control group throughout the study. At or close to ovulation, the fecundity of ULO fish was 75-80% that of controls. In the control group, oocytes appeared to reach a certain critical size before they were ovulated, and fish with higher fecundity ovulated later than their less fecund counterparts. ULO did not affect the timing of ovulation, and ULO fish ovulated eggs with a considerably greater size-range than did controls.