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There is growing evidence that rising global temperatures resulting from climate change may exacerbate the toxic effect of pollutants and heterotherms, including fish, in which homestatic mechanisms are directly influenced by environmental temperature will be most affected. Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many of their drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity but this has received little consideration in the context of effects in wildlife. Further, these mechanisms are relatively poorly understood, particularly regarding multiple stressor interactions. We used transgenic TG(EpRE:mCherry) zebrafish, developed in our laboratory for detecting OS, as our experimental model. We show that the oxidative effects of high concentrations of pharmaceuticals from three different therapeutic classes (paracetamol, diclofenac and doxorubicin) are increased at temperatures elevated by 2-5 °C above those for zebrafish standard husbandry and relevant to their current natural environment (and predicted under the IPCC 2023 scenarios for intermediate to very high greenhouse gas emissions). These OS responses were primarily seen in the pronephros, liver, and gastrointestinal tract. The increase in OS at the increased water temperature may have resulted from the elevated temperature acting as a direct additive physiological stressor to the OS imposed by the drugs and/or via the temperature increasing the chemicals oxidative effect. For paracetamol, it appeared that the elevated responses at the higher temperature of 33 °C were in part due to an increase in uptake of the drug. Our data illustrate that risk assessments for chemicals inducing OS in fish (and likely other heterotherms) should consider the influence of temperature to ensure environmental protection in future environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures., Competing Interests: Declaration of Competing Interest SFO is an employee and shareholder of AstraZeneca PLC. All other authors declare no potentially competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Globally, millions of zebrafish (Danio rerio) are used for scientific laboratory experiments for which researchers have a duty of care, with legal obligations to consider their welfare. Considering the growing use of the zebrafish as a vertebrate model for addressing a diverse range of scientific questions, optimising their laboratory conditions is of major importance for both welfare and improving scientific research. However, most guidelines for the care and breeding of zebrafish for research are concerned primarily with maximising production and minimising costs and pay little attention to the effects on welfare of the environments in which the fish are maintained, or how those conditions affect their scientific research. Here we review the physical and social conditions in which laboratory zebrafish are kept, identifying and drawing attention to factors likely to affect their welfare and experimental science. We also identify a fundamental lack knowledge of how zebrafish interact with many biotic and abiotic features in their natural environment to support ways to optimise zebrafish health and well-being in the laboratory, and in turn the quality of scientific data produced. We advocate that the conditions under which zebrafish are maintained need to become a more integral part of research and that we understand more fully how they influence experimental outcome and in turn interpretations of the data generated., (© 2022 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

We assessed similarities and differences in the electrographic signatures of local field potentials (LFPs) evoked by different pharmacological agents in zebrafish larvae. We then compared and contrasted these characteristics with what is known from electrophysiological studies of seizures and epilepsy in mammals, including humans. Ultimately, our aim was to phenotype neurophysiological features of drug-induced seizures in larval zebrafish for expanding knowledge on the translational potential of this valuable alternative to mammalian models. LFPs were recorded from the midbrain of 4-d-old zebrafish larvae exposed to a pharmacologically diverse panel of seizurogenic compounds, and the outputs of these recordings were assessed using frequency domain analysis. This included analysis of changes occurring within various spectral frequency bands of relevance to mammalian CNS circuit pathophysiology. From these analyses, there were clear differences in the frequency spectra of drug-exposed LFPs, relative to controls, many of which shared notable similarities with the signatures exhibited by mammalian CNS circuits. These similarities included the presence of specific frequency components comparable to those observed in mammalian studies of seizures and epilepsy. Collectively, the data presented provide important information to support the value of larval zebrafish as an alternative model for the study of seizures and epilepsy. These data also provide further insight into the electrophysiological characteristics of seizures generated in nonmammalian species by the action of neuroactive drugs., (Copyright © 2022 Pinion et al.)

Background and Purpose: Functional brain imaging using genetically encoded Ca 2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABA A antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy., Experimental Approach: Using light-sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link., Key Results: We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic consistency broadly mapping onto what is known about neurotransmitter-specific circuitry in the larval zebrafish brain. Many seizure-associated compounds also exhibited altered whole brain functional connectivity compared with controls., Conclusions and Implications: This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small-scale pharmacological activation of local circuits, to the large network-wide abnormal synchronous activity associated with seizures., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin ( vtg ) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish's developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cooper, David, Lange and Tyler.)

Background: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity., Objectives: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS., Methods: To achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity., Results: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity., Discussion: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer., (Copyright © 2019. Published by Elsevier Ltd.)

Brominated flame retardants are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, but the molecular mechanisms underlying this process and their effects on TH-sensitive tissues during the stages of early development are not well characterised. In this study, we exposed zebrafish (Danio rerio) embryo-larvae to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and tetrabromobisphenol A (TBBPA) via the water for 96 h from fertilisation and assessed for lethality, effects on development and on the expression of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis via both real time quantitative PCR (qRT-PCR) on whole body extracts and whole mount in situ hybridisation (WISH) to identify tissue targets. The 96-h lethal median concentration (96h-LC 50 ) for TBBPA was 0.9 μM and mortality was preceded by retardation of development (smaller animals) and morphological deformities including, oedemas in the pericardial region and tail, small heads, swollen yolk sac extension. Exposure to BDE-47 did not affect zebrafish embryo-larvae survival at any of the concentrations tested (1-100 μM) but caused yolk sac and craniofacial deformities, a curved spine and shorter tail at the highest exposure concentration. TBBPA exposure resulted in higher levels of mRNAs for genes encoding deiodinases (dio1), transport proteins (ttr), the thyroid follicle synthesis protein paired box 8 (pax8) and glucuronidation enzymes (ugt1ab) and lower levels of dio3b mRNAs in whole body extracts, with responses varying with developmental stage. BDE-47 exposure resulted in higher levels of thrb, dio1, dio2, pax8 and ugt1ab mRNAs and lower levels of ttr mRNAs in whole body extracts. TBBPA and BDE-47 therefore appear to disrupt the TH system at multiple levels, increasing TH conjugation and clearance, disrupting thyroid follicle development and altering TH transport. Compensatory responses in TH production/ metabolism by deiodinases were also evident. WISH analyses further revealed that both TBBPA and BDE-47 caused tissue-specific changes in thyroid receptor and deiodinase enzyme expression, with the brain, liver, pronephric ducts and craniofacial tissues appearing particularly responsive to altered TH signalling. Given the important role of TRs in mediating the actions of THs during key developmental processes and deiodinases in the control of peripheral TH levels, these transcriptional alterations may have implications for TH sensitive target genes involved in brain and skeletal development. These findings further highlight the potential vulnerability of the thyroid system to disruption by BFRs during early developmental windows., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

Environmental enrichment involves increasing the complexity of a fish's environment in order to improve welfare. Researchers are legally obliged to consider the welfare of laboratory animals and poor welfare may result in less robust data in experimental science. Laboratory zebrafish Danio rerio are usually kept in bare aquaria for ease of husbandry and, despite being a well-studied species, little is known about how laboratory housing affects their welfare. This study shows that environmental enrichment, in the form of the addition of gravel substratum and plants into the tank, affects survivorship, growth and behaviour in laboratory-maintained D. rerio. Larvae reared in enriched tanks had significantly higher survivorship compared with larvae reared in bare tanks. Effects of the tank conditions on growth were more variable. Females from enriched tanks had a higher body condition than females maintained in bare tanks, but intriguingly this was not the case for males, where the only difference was a more variable body condition in males maintained in bare tanks. Sex ratio in the rearing tanks did not differ between treatments. Resource monopolisation was higher for fish in enriched tanks than for those in bare tanks. Fish from enriched tanks displayed lower levels of behaviours associated with anxiety compared with fish from bare tanks when placed into a novel environment. Thus, this study demonstrates differences in welfare for D. rerio maintained under different environmental conditions with enhancements in welfare more commonly associated with tank enrichment., (© 2018 The Fisheries Society of the British Isles.)

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

This study examined the effects of simple changes in the tank environment on the wellbeing of laboratory-maintained zebrafish Danio rerio. Groups of D. rerio were either housed in stable environments (where they were maintained in the same tanks throughout the study) or in environments subject to change (where they were periodically moved to novel but identical tanks) and the effects of these treatments on morphometry, reproductive success and aggressive behaviour assessed. No effect of simple tank changes was found on body condition, reproductive output or aggression, for the periods of time studied, indicating that more complex scenarios in housing tank conditions are required for significant welfare benefits for captive D. rerio., (© 2018 The Fisheries Society of the British Isles.)

Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

Copper nanoparticles (CuNPs) are used extensively in a wide range of products and the potential for toxicological impacts in the aquatic environment is of high concern. In this study, the fate and the acute toxicity of spherical 50nm copper nanoparticles was assessed in juvenile rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas) and zebrafish (Danio rerio) for in vivo aqueous exposures following standardized OECD 203 guideline tests. The fate of the CuNPs in the aqueous media was temperature dependent. At the higher study temperature (26±1°C), there was both an enhanced particle aggregation and higher rate of dissolution compared with that at the lower study temperature (15±1°C). 96h LC50s of the CuNPs were 0.68±0.15, 0.28±0.04 and 0.22±0.08mg Cu/L for rainbow trout, fathead minnow and zebrafish, respectively. The 96h lowest-observed-effect concentration (LOEC) for the CuNPs were 0.17, 0.023 and <0.023mg/L for rainbow trout, fathead minnow, and zebrafish respectively, and are below the predicted environmental concentration of CuNPs for some aquatic environments suggesting a possible ecotoxicological risk to fish. Soluble copper was one of main drivers for the acute toxicity of the copper nanoparticles suspensions. Both CuNPs suspension and copper nitrate caused damage to gill filaments and gill pavement cells, with differences in sensitivity for these effects between the fish species studied. We show therefore common toxicological effects of CuNPs in different fish species but with differences in sensitivity with implications for hazard extrapolation between fish species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Endocrine disrupting chemicals (EDCs) are potent environmental contaminants, and their effects on wildlife populations could be exacerbated by climate change, especially in species with environmental sex determination. Endangered species may be particularly at risk because inbreeding depression and stochastic fluctuations in male and female numbers are often observed in the small populations that typify these taxa. Here, we assessed the interactive effects of water temperature and EDC exposure on sexual development and population viability of inbred and outbred zebrafish (Danio rerio). Water temperatures adopted were 28 °C (current ambient mean spawning temperature) and 33 °C (projected for the year 2100). The EDC selected was clotrimazole (at 2 μg/L and 10 μg/L), a widely used antifungal chemical that inhibits a key steroidogenic enzyme [cytochrome P450(CYP19) aromatase] required for estrogen synthesis in vertebrates. Elevated water temperature and clotrimazole exposure independently induced male-skewed sex ratios, and the effects of clotrimazole were greater at the higher temperature. Male sex ratio skews also occurred for the lower clotrimazole exposure concentration at the higher water temperature in inbred fish but not in outbred fish. Population viability analysis showed that population growth rates declined sharply in response to male skews and declines for inbred populations occurred at lower male skews than for outbred populations. These results indicate that elevated temperature associated with climate change can amplify the effects of EDCs and these effects are likely to be most acute in small, inbred populations exhibiting environmental sex determination and/or differentiation.

The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4-UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities.

The vas::egfp transgenic zebrafish (Danio rerio) could significantly enhance studies on the mechanisms by which environmental estrogens disrupt sexual differentiation, because the developing gonad can be visualized during early life via fluorescence detection. There are methodological challenges regarding dissecting out the gonads in early-life-stage fish, however, and transgene responses to estrogen exposure have not been tested. The authors exposed vas::egfp transgenic zebrafish and their wild-type siblings to the model estrogen 17α-ethinylestradiol (EE2; 0.62 ng/L and 3.33 ng/L measured concentrations) during sexual development (20-60 d posthatch) and used enhanced green fluorescent protein (egfp) fluorescence to identify and dissect single gonads (at 40 d posthatch) to provide sufficient RNA for individual gene expression analyses, retaining the remaining gonad in the body cavity for histological analyses of sex and stage of development. Genotyping confirmed that all transgenic control fish were phenotypically egfp positive (showed green fluorescence). Interestingly, however, in a few transgenic fish exposed to EE2, no phenotypic egfp signal was seen, most notably for the 3.33 ng/L EE2 exposure. It was subsequently found that gonadal vasa expression was reduced by this concentration of EE2. Hepatic vitellogenin expression demonstrated that the vas::egfp and wild-type lines responded to estrogen with an equivalent sensitivity. The authors conclude that the vas::egfp zebrafish provides an enhanced and practical system for mechanistic studies on the effects of environmentally relevant concentrations of estrogens on gonad development., (© 2013 SETAC.)

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.

Genetic variation has a significant effect on behavior, fitness, and response to toxicants; however, this is rarely considered in ecotoxicological studies. We compared fitness-related behavioral traits, breeding activity, and the effects of exposure to the environmental estrogen ethinylestradiol (EE(2)) on reproduction in a laboratory (Wild Indian Karyotype, WIK) strain and a wild-caught population (Bangladesh, BLD01) of Danio rerio (zebrafish). In WIK fish, males with higher observed heterozygocity were more active reproductively and more successful in securing parentage, but these relationships were not apparent in the BLD01 fish. The frequency of reproductive behaviors increased in WIK zebrafish for exposure to 0.4 ng/L EE(2), which was not apparent in the BLD01 zebrafish. The different strains showed the same threshold for hepatic vitellogenin gene (vtg) induction (2.2 ng EE(2)/L), but results suggested an elevated response level in the BLD01. There were no effects on total egg production up to 2.2 ng EE(2)/L in either population; however, there was reduced egg fertilization rate at 2.2 ng EE(2)/L in the BLD01 fish. These results show consistency in the general responses to EE(2) between these two genetically divergent strains of zebrafish, but also illustrate differences in their breeding biology and response sensitivities. These findings highlight the need for due consideration of the source (and genetics) of populations used in ecological risk assessment for accurate comparisons among studies.

Background: Environmental estrogens alter hormone signaling in the body that can induce reproductive abnormalities in both humans and wildlife. Available testing systems for estrogens are focused on specific systems such as reproduction. Crucially, however, the potential for significant health impacts of environmental estrogen exposures on a variety of body systems may have been overlooked., Objective: Our aim was to develop and apply a sensitive transgenic zebrafish model to assess real-time effects of environmental estrogens on signaling mechanisms in a whole body system for use in integrated health assessments., Methods: We created a novel transgenic biosensor zebrafish containing an estrogen-inducible promoter derived with multiple tandem estrogen responsive elements (EREs) and a Gal4ff-UAS system for enhanced response sensitivity., Results: Using our novel estrogen-responsive transgenic (TG) zebrafish, we identified target tissues for environmental estrogens; these tissues have very high sensitivity even at environmentally relevant concentrations. Exposure of the TG fish to estrogenic endocrine-disrupting chemicals (EDCs) induced specific expression of green fluorescent protein (GFP) in a wide variety of tissues including the liver, heart, skeletal muscle, otic vesicle, forebrain, lateral line, and ganglions, most of which have not been established previously as targets for estrogens in fish. Furthermore, we found that different EDCs induced GFP expression with different tissue response patterns and time trajectories, suggesting different potential health effects., Conclusion: We have developed a powerful new model for understanding toxicological effects, mechanisms, and health impacts of environmental estrogens in vertebrates.

Outbred laboratory animal strains used in ecotoxicology are intended to represent wild populations. However, breeding history may vary considerably between strains, driving differences in genetic variation and phenotypes used for assessing effects of chemical exposure. We compared a range of phenotypic endpoints in zebrafish from four different "breeding treatments" comprising a Wild Indian Karyotype (WIK) zebrafish strain and a WIK/Wild strain with three levels of inbreeding (F(IT)=n, n+0.25, n+0.375) in a new Fish Sexual Development Test (FSDT). There were no differences between treatments in terms of egg viability, hatch success or fry survival. However, compared with WIKs, WIK/Wild hybrids were significantly larger in size, with more advanced gonadal (germ cell) development at the end of the test (63 days post fertilisation). Increasing the levels of inbreeding in the related WIK/Wild lines did not affect body size, but there was a significant male-bias (72%) in the most inbred line (F(IT)=n+0.375). Conversely, in the reference WIK strain there was a significant female-bias in the population (80% females). Overall, our results support the use of outbred zebrafish strains in the FSDT, where one of the core endpoints is sex ratio. Despite increased variance (and reduced statistical power) for some endpoints, WIK/Wild outbreds (F(IT)=n) met all acceptance criteria for controls in this test, whereas WIKs failed to comply with tolerance limits for sex ratio (30-70% females). Sexual development was also more advanced in WIK/Wild outbreds (cf. WIKs), providing greater scope for detection of developmental reproductive toxicity following chemical exposure., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Environmental estrogens have been shown to affect aspects of fish behavior that could potentially impact on wild populations, but the physiological mechanisms underpinning these effects are unknown. Using small colonies of zebrafish (Danio rerio), we evaluated the impacts of estrogen exposure on the aggression of dominant males, the associated implications for their social status and reproductive success, and their signaling mechanisms. The aggression of dominant males exposed to 17α-ethinylestradiol (EE(2); 10 ng/L nominal) was reduced significantly, and half of these fish subsequently lost their dominance, behavioral changes that were reflected in their reproductive success. Plasma androgen and the expression of genes involved in sex steroid production/signaling (cyp19a1b, cyp17, hsd11b2, hsd17b3, ar) and aggression (avplrv1b, tph1b, htr1a, sst1, sstr1, th, slc6a3, ar) were higher in control dominant versus subordinate males, but suppressed by EE(2) exposure, such that the differences between the social ranks were not retained. The expression levels of avpl (brain), which promotes aggression and dominance, and ar and cyp17 (gonad) were elevated in nonexposed males paired with EE(2)-exposed males. Our findings illustrate that disruptions of behaviors affecting social hierarchy, and in turn breeding outcome, as a consequence of exposure to an environmental estrogen are signaled through complex interconnecting gonadal and neurological control mechanisms that generally conform with those established in mammalian models. The extensive molecular, genetic, physiological, and behavioral toolbox now available for the zebrafish makes this species an attractive model for integrated analyses of chemical effects spanning behavior to molecular effect mechanisms.

Population regulation is fundamental to the long-term persistence of populations and their responses to harvesting, habitat modification, and exposure to toxic chemicals. In fish and other organisms with complex life histories, regulation may involve density dependence in different life-stages and vital rates. We studied density dependence in body growth and mortality through the life-cycle of laboratory populations of zebrafish Danio rerio. When feed input was held constant at population-level (leading to resource limitation), body growth was strongly density-dependent in the late juvenile and adult phases of the life-cycle. Density dependence in mortality was strong during the early juvenile phase but declined thereafter and virtually ceased prior to maturation. Provision of feed in proportion to individual requirements (easing resource limitation) removed density dependence in growth and substantially reduced density dependence in mortality, thus indicating that 'bottom-up' effects act on growth as well as mortality, but most strongly on growth. Both growth and mortality played an important role in population regulation, with density-dependent growth having the greater impact on population biomass while mortality had the greatest impact on numbers. We demonstrate a clear ontogenic pattern of change in density-dependent processes within populations of a very small (maximum length 5 mm) fish, maintained in constant homogeneous laboratory conditions. The patterns are consistent with those distilled from studies on wild fish populations, indicating the presence of broad ontogenic patterns in density-dependent processes that are invariant to maximum body size and hold in homogeneous laboratory, as well as complex natural environments.

Laboratory animals tend to be more inbred and less genetically diverse than wild populations, and thus may differ in their susceptibility to chemical stressors. We tested this hypothesis by comparing the responses of related inbred (theoretical inbreeding F(IT) = n + 0.25) and outbred (F(IT) = n) zebrafish (Danio rerio) WIK/Wild family lines to an endocrine disrupting chemical, clotrimazole. Exposure of inbred and outbred zebrafish to 2.9 μg clotrimazole/L had no effect on survival, growth, or gonadal development. Exposure of both lines to 43.7 μg clotrimazole/L led to male-biased sex ratios compared with controls (87% versus 55% and 92% vs 64%, for inbred and outbred males, respectively), advanced germ cell development, and reduced plasma 11-ketotestosterone concentrations in males. However, outbred males (but not inbred males) developed testis that were more than twice the weight of controls, which corresponded with a proliferation of Leydig cells and maintenance of the expression (rather than down-regulation occurring in inbreds) of gonadal aromatase (cyp19a1a) and insulin-like growth factor (igf1). Our results illustrate that the effects of an endocrine disrupting chemical (clotrimazole) on some end points (here testis development) can differ between inbred and outbred zebrafish. This highlights the need for reporting pedigree/genetic information and consistency in the responses of laboratory animals (e.g., by using model compounds as positive controls).

Social status affects access to food, mates and shelter and has consequences for the physiology of individuals and their health status. In the zebrafish (Danio rerio), an emerging model for studies into animal behavior, the possible consequences of social hierarchy to an individual's physiology and health are unknown. To address this, in this species we assessed the effects of social interaction (for periods of 1-5days) on growth, stress, immune function and reproductive condition. Wide-ranging differences in physiology occurred between the social ranks, some of which were sex-related and time-dependent. In both sexes, dominant fish were larger than subordinates and dominant males had a higher growth rate during the trials. Subordinates had higher plasma cortisol and in males higher telencephalic corticotrophin-releasing hormone, neuropeptide y and glucocorticoid receptor gene expression. Splenic cytokine expression suggested differences in immune status between ranks in both sexes and hematocrit was elevated in subordinate males. In both sexes, dominants and subordinates differed in the expression of genes for various gonadal sex steroid receptors and steroidogenic enzymes and in dominant females the ovary was larger relative to body mass compared with in subordinates. Dominant males had higher plasma 11-ketotestosterone than subordinates and there was an increase in the number of spermatids in their testes over the duration of the study that was not seen in subordinate males. The wide-ranging physiological differences seen between dominant and subordinate zebrafish as a consequence of their social status suggest negative health impacts for subordinates after prolonged durations in those hierarchies., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish (Danio rerio), an animal model in which social rank and aggressiveness tightly correlate., Results: Substantial differences occurred in gene expression profiles between dominant and subordinate males associated with phenotypic differences in aggressiveness and, for the chosen gene set, they occurred mainly in the hypothalamus and telencephalon. The patterns of differentially-expressed genes implied multifactorial control of aggression in zebrafish, including the hypothalamo-neurohypophysial-system, serotonin, somatostatin, dopamine, hypothalamo-pituitary-interrenal, hypothalamo-pituitary-gonadal and histamine pathways, and the latter is a novel finding outside mammals. Pharmacological manipulations of various nodes within the hypothalamo-neurohypophysial-system and serotonin pathways supported their functional involvement. We also observed differences in expression profiles in the brains of dominant versus subordinate females that suggested sex-conserved control of aggression. For example, in the HNS pathway, the gene encoding arginine vasotocin (AVT), previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors., Conclusions: Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model.

Impacts of exposure to environmental estrogens on reproductive development are well documented, but recently wider concern has been raised due to evidence that such exposures can disrupt normal patterns of reproductive behavior, dominance, and parentage, with potential population level implications. It is fundamental therefore to understand any such effects for effective risk assessment. This study investigated the impact of a transient exposure to ethinylestradiol (EE(2)) during early life (from 20-60 days post fertilization), including at a dosing level within the environmental range, on the subsequent reproductive behavior and success in both male and female zebrafish (Danio rerio) in competitive breeding scenarios. There were no obvious effects of the early life EE(2) exposures on the subsequent gonadal phenotypes in either mature males or females. In fact, reproductive success in males exposed to 2.76 ng EE(2)/L was increased in competitive spawning scenarios. In contrast, exposure of females to EE(2) (9.86 ng/L) during early life reduced their subsequent reproductive success in competitive spawning scenarios. Mate choice experiments suggested this was a consequence of the females' diminished courting behavior toward males, rather than any male preference for unexposed females. Reproductive capability of females is generally considered a key determinant in population demographics and dynamics, and therefore the effect of exposure to EE(2) on female reproductive success may have significant implications for exposed fish populations.

The zebrafish has considerable potential for use as a model in the study of behavior in social systems, particularly dominance hierarchies, which are widespread in nature and can affect the lifelong success of individuals. There is, however, a paucity of information relating to the characterization of social groups and significance of dominance hierarchies in the zebrafish model. This study set out to bridge this knowledge gap and better characterize dominance and its implications for reproductive success in both male and female zebrafish in colonies comprising of two males and two females. Analyses of four aggressive behaviors (chase, bite, repel, spar) were conducted twice daily over a 5-day period, and fertilized eggs were collected for parentage analyses using DNA microsatellite markers. Dominant-subordinate relationships occurred both between males and between females, and in both sexes, dominance was associated with a greater body size and higher levels of aggression. During the spawning period, dominant females were, however, less aggressive toward their subordinates than dominant males to their subordinates. Aggressive behaviors employed for maintaining dominance did not differ between the sexes, but in females, in contrast with males, the level of aggression directed toward the subordinate fish increased over the study period. Overall, dominance resulted in a greater total reproductive success in males but not in females; however, dominant females sired more offspring with the dominant male. The findings illustrate that energy invested in dominance behavior appears beneficial for both sexes in zebrafish.

Evidence has recently emerged that endocrine-disrupting chemicals (EDCs) can affect various behaviors, including dominance and aggression in social groups, including fish. This study investigated the effect of short-term exposure of male adult zebrafish to 17alpha-ethinylestradiol (EE(2)) on subsequent reproductive output and parentage in colonies with differing numbers of competing males. It was predicted that impacts of EDCs might differ in social groups of fish of differing size because of the greater costs of maintaining dominance hierarchies in large groups. Adult male zebrafish were exposed for 14 days to clean water, 2 ng/L EE(2) or 10 ng/L via the water, prior to placement into colonies in clean water with unexposed females. Exposure to EE(2) at the concentrations adopted prior to the breeding trials did not significantly affect subsequent colony reproductive output. The reproductive success of the most reproductively successful (MRS) male within colonies containing two males (relative to controls) was also unaffected. There was, however, a significant impact of previous EE(2) exposure in tanks containing four males, resulting in a reduction in paternity for the most successful male. Hence, nonlethal behavioral impacts of even short-term exposure to EDCs can have significant impacts on social dominance hierarchies and population genetic diversity.

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 l(-1) to microg l(-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.

There is substantial evidence that genetic variation, at both the level of the individual and population, has a significant effect on behaviour, fitness and response to toxicants. Using DNA microsatellites, we examined the genetic variation in samples of several commonly used laboratory strains of zebrafish, Danio rerio, a model species in toxicological studies. We compared the genetic variation to that found in a sample of wild fish from Bangladesh. Our findings show that the wild fish were significantly more variable than the laboratory strains for several measures of genetic variability, including allelic richness and expected heterozygosity. This lack of variation should be given due consideration for any study which attempts to extrapolate the results of ecotoxicological laboratory tests to wild populations.

There is global concern regarding the potential impacts of endocrine-disrupting chemicals (EDCs) on the health of wildlife and humans. Exposure to some estrogens, at concentrations found in the environment impairs reproductive function and behavior. However, nearly all work on endocrine disruption has investigated the effects of exposure on individuals and there is an urgent need to understand impacts on populations. Many fish have mating systems with complex social structures and it is not known whether EDCs will exaggerate or buffer the reproductive skews caused by the dominance hierarchies that normally occur for these species. This study investigated the impact of exposure to the pharmaceutical estrogen ethinylestradiol (EE2) on reproductive hierarchies and sexual selection in group-spawning fish. Breeding zebrafish were exposed to environmentally relevant concentrations of EE2, and effects were determined on reproductive output, plasma androgen concentrations (in males), and reproductive success through microsatellite analyses of the offspring. Reproductive hierarchies in breeding colonies of zebrafish were disrupted by exposure to EE2 at a concentration that did not affect the number of eggs produced. The effect was a reduction in the skew in male paternity and increased skew in female maternity. This disruption in the reproductive hierarchy in group spawning fish, if it occurs in the wild, has potentially major implications for population genetic diversity. Reproductive success in male zebrafish was associated with elevated plasma concentrations of the male sex hormone 11-ketotestosterone and this hormone was suppressed in EE2-exposed males.

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.

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.

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.

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.

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.

Heightened concern over endocrine-disrupting chemicals is driven by the hypothesis that they could reduce reproductive success and affect wildlife populations, but there is little evidence for this expectation. The pharmaceutical ethynylestradiol (EE2) is a potent endocrine modulator and is present in the aquatic environment at biologically active concentrations. To investigate impacts on reproductive success and mechanisms of disruption, we exposed breeding populations (n = 12) of zebrafish (Danio rerio) over multiple generations to environmentally relevant concentrations of EE2. Life-long exposure to 5 ng/L EE2 in the F1 generation caused a 56% reduction in fecundity and complete population failure with no fertilization. Conversely, the same level of exposure for up to 40 days in mature adults in the parental F0 generation had no impact on reproductive success. Infertility in the F1 generation after life-long exposure to 5 ng/L EE2 was due to disturbed sexual differentiation, with males having no functional testes and either undifferentiated or intersex gonads. These F1 males also showed a reduced vitellogenic response when compared with F0 males, indicating an acclimation to EE2 exposure. Depuration studies found only a partial recovery in reproductive capacity after 5 months. Significantly, even though the F1 males lacked functional testes, they showed male-pattern reproductive behavior, inducing the spawning act and competing with healthy males to disrupt fertilization. Endocrine disruption is therefore likely to affect breeding dynamics and reproductive success in group-spawning fish. Our findings raise major concerns about the population-level impacts for wildlife of long-term exposure to low concentrations of estrogenic endocrine disruptors.

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17alpha-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30-85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5+/-2.7 and 4.9+/-1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) < or =1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.