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.

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.

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.

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.

The ornamental fish trade is an industry of significant size and scope, trading over 1.5 billion fishes each year, and worth approximately over 370 million USD. Ornamental fishes are kept world-wide, and are one of the most popular pets in UK households. The industry is currently experiencing steady growth, and has done since the FAO began keeping records in the 1970s. Despite this, the welfare of fishes within the industry remains one of the least-studied areas in the field of animal welfare. Mortality rates of fishes within the industry are debated, with estimates ranging from less than 2% to over 70%; however, a lack of clear data means that the accuracy of these figures is difficult to determine. Where mortality is believed to be high, stressors in the supply chain are thought to be a significant contributing factor. In this thesis, I explored some possible interventions designed to reduce the stress experienced by ornamental fishes.
Stress in fishes can be measured in a variety of ways, but the most common way is probably measurement of cortisol release rates. However, cortisol has often previously been measured in fishes by taking a blood sample – a technique which cannot be applied to many ornamental species as they are too small to obtain enough blood. Instead, cortisol released by small fishes can be measured in the fish holding water. I carried out a study to validate the use of this method in my study species and found that cortisol can be detected in the holding water of all three species, although I did not find clear differences between stressed fishes and controls. This highlighted the importance of using a variety of measures of stress, including behavioural measures, which are one of the most cost-effective ways to assess stress, and can easily be implemented in the ornamental fish supply chain.
Based on the literature, personal observations of industry practices, and the results of my analyses, a number of interventions intended to help reduce stress in ornamental species were developed. These involved training handlers to catch fish more effectively, providing neon tetras with environmental choices to allow them to select conditions which might promote welfare, and conditioning guppies to associate handling events with a reward or a predictable signal. I found fish which were not handled but were exposed to trained handlers showed fewer behavioural signs of stress than those exposed to untrained handlers, and that neon tetras showed preferences for particular tank backgrounds over others. However, I did not find any evidence that trained handlers caused less stress in handled fish, or that conditioning led to lower stress in handled fish.
the results of this project suggest that there are a number of sources of stress and poor welfare in the ornamental fish industry which may be contributing to high mortality rates. However, many of these sources can be addressed, either through application of current best-practice guidelines or by introduction of training programmes which encourage understanding and empathy for fishes. Further work aimed at developing interventions including enrichment strategies, conditioning regimes, and other areas of research, will likely help to further reduce stress and mortality, and improve fish welfare.

The ornamental fish trade is estimated to handle up to 1·5 billion fishes. Transportation and handling of fishes imposes a range of stressors that can result in mortality at rates of up to 73%. These rates vary hugely, however, and can be as low as 2%, because they are generally estimated rather than based on experimental work. Given the numbers of ornamental fishes traded, any of the estimated mortality rates potentially incur significant financial losses and serious welfare issues. Industry bodies, such as the Ornamental Aquatic Trade Association (OATA), have established standards and codes of best practice for handling fishes, but little scientific research has been conducted to understand the links between stress, health and welfare in ornamental species. In aquaculture, many of the same stressors occur as those in the ornamental trade, including poor water quality, handling, transportation, confinement, poor social and physical environment and disease and in this sector directed research and some resulting interventions have resulted in improved welfare standards. This review considers the concept of welfare in fishes and evaluates reported rates of mortality in the ornamental trade. It assesses how the stress response can be quantified and used as a welfare indicator in fishes. It then analyses whether lessons from aquaculture can be usefully applied to the ornamental fish industry to improve welfare. Finally, this analysis is used to suggest how future research might be directed to help improve welfare in the ornamental trade.

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.

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.

Inbreeding depression is expected to be more severe in stressful environments. However, the extent to which inbreeding affects the vulnerability of populations to environmental stressors, such as chemical exposure, remains unresolved. Here we report on the combined impacts of inbreeding and exposure to an endocrine disrupting chemical (the fungicide clotrimazole) on zebrafish (Danio rerio). We show that whilst inbreeding can negatively affect reproductive traits, not all traits are affected equally. Inbreeding depression frequently only became apparent when fish were additionally stressed by chemical exposure. Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring when in direct competition with outbred individuals. Levels of plasma 11-ketotestosterone, a key male sex hormone, showed substantial inbreeding depression that was unaffected by addition of the fungicide. In contrast, there was no effect of inbreeding or clotrimazole exposure on egg production. Overall, our data provide evidence that stress may amplify the effects of inbreeding on key reproductive traits, particularly those associated with male fitness. This may have important implications when considering the consequences of exposure to chemical pollutants on the fitness of wild populations. Journal compilation © 2013 Blackwell Publishing Ltd62 February 2013 10.1111/j.1752-4571.2012.00288.x Original Article Original Articles © 2012 Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution Non Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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.

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.

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).

Feminized responses are widespread in wild populations of roach, Rutilus rutilus, living in UK rivers, and some of these responses have been shown to arise as a consequence of exposure to wastewater treatment works (WwTW) effluent discharges and the endocrine disrupting chemicals (EDCs) they contain. The causation of the ovotestis condition in wild roach, however, has yet to be established. Furthermore, the impact of long-term exposure to WwTW effluents on the reproductive fitness of wild fish populations is not known, and this information is crucial for population level effect assessments. We undertook a chronic exposure of roach to a treated estrogenic wastewater effluent for up to 3.5 years to assess principally for effects on subsequent reproductive fitness, as determined through parentage analysis on offspring from a competitive breeding study. In generating the fish for the breeding study we found that exposure to full strength WwTW effluent until sexual maturity resulted in sex reversal in almost all males in the population; 98% of the exposed fish were phenotypic females, containing ovaries. Furthermore, fish exposed to a 50% dilution of WwTW effluent contained ovotestis (21% of the male roach) that was absent from the control population. In competitive breeding studies, and applying DNA microsatellites to assess parentage, we show that presumptive females exposed to sexual maturity to WwTW effluent bred normally, albeit in the absence of nonexposed females, but putative sex-reversed males breeding as females contributed poorly, if at all, in a breeding population, depending on the competition. These novel findings on sex reversal add a new dimension for impact assessments of exposure to WwTW effluents on 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.

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.

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.

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.

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.

Many environmental factors have been shown to influence sex differentiation in fish, resulting in sex-biased populations, but the effects of growth rate have received limited attention. We conducted a controlled laboratory experiment in which growth rate and population density were manipulated in roach (Rutilus rutilus) during early development, and the subsequent effects on sex ratio determined. Significant differences in growth rate between fish populations were induced through provision of three different ration levels. In the slowest growing population there were fewer females compared within the fastest growing population (19% compared to 36% females), suggesting that in roach it may be more advantageous to become a small male than a small female when growth potential is limited. This may result from the fact that fecundity is limited by body size in female roach and that male roach are able to reproduce at a significantly smaller body size than females. In contrast, where roach were kept at different stocking densities, and there were no differences in growth rate, the subsequent proportion of females did not vary. Our data highlight the importance of controlling for growth rate in research on sexual differentiation in this species, notably when assessing for the effects of endocrine disrupting chemicals and other environmental factors, and have implications for fisheries management and aquaculture. The underlying mechanism for the influence of growth rate on sex differentiation has yet to be determined but is likely to have a strong endocrinological basis. © Springer Science+Business Media B.V. 2009.

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.

Globally, feminization responses in wild male freshwater fish are caused by exposure to estrogenic chemicals, including natural and synthetic estrogens, contained in effluentsfromwastewater treatment works. In U.K. rivers, feminization responses, including intersex, are widespread in wild roach (Rutilus rutilus) populations, and severely affected fish have a reduced reproductive success. We exposed roach to environmentally relevant concentrations of the contraceptive estrogen 17alpha-ethinylestradiol (EE2) for up to 2 years, including intermittent and repeated exposures,to determine effects on sexual development and subsequent responsiveness to estrogen. Exposure of roach to EE2 (at 4 ng/L) for 2 years resulted in sex reversal in males, leading to an all-female population with two cohorts in terms of their stages of ovarian development one paralleling the control females and one at a significantly less advanced stage, which we propose were sex-reversed males. Differing developmental and maturing rates of the putative sex-reversed males compared with control females would question their functional capability as females in the wild. Early-life exposure to environmentally relevant concentrations of EE2 sensitized females to estrogen, as determined by the measurement of the responses of estrogen-sensitive genes in a further EE2 challenge 398 days after the original exposure. In the wild, exposure to environmentally relevant concentrations of EE2 during early life has significantly wider implications for the sexual physiology in fish than has thus far been determined.

Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17alpha-ethinylestradiol (EE(2)) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE(2) during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development.

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.

The roach (Rutilus rutilus) has become a sentinel species for the study of sexual disruption in wild fish populations as a consequence of exposure to endocrine disrupting chemicals (EDCs). Little is known, however, about the normal ontogeny of sexual development in this species. Here, we analyzed the ontogeny of sexual development in captive-bred roach and assessed how growth rate and fish size affected the timing of both sexual differentiation and sexual development over a 2-year period. Ovarian differentiation was first recorded at 68 days post-fertilization (dpf) and this preceded testicular differentiation (first recorded at 98 dpf). In contrast, sexual maturation occurred at an earlier age in males (300 dpf) compared with females (728 dpf). No differences in body size (length or weight) were recorded between male and female roach until the fish were 415 dpf. Studies on three populations of roach which grew at different rates showed that the timing of sexual differentiation was highly variable and more related to fish size than to fish age. Time to sexual maturation was also variable among populations but, subsequent to their first year of life, gonadal status was less well associated with fish size. Interestingly, the sex ratio of the population was biased towards females in populations that grew more rapidly during early life. The findings presented here provide a valuable foundation of work to support both field- and laboratory-based assessments on the effects of EDCs, and other stressors, on sexual differentiation and development in the roach.

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.

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.

Alterations in development and reproduction as a consequence of exposure to endocrine-disrupting chemicals (EDCs) have been demonstrated in many wildlife species. Animals living in, or closely associated with, the aquatic environment are particularly vulnerable to endocrine disruption because thousands of chemicals are actively disposed into rivers, estuaries and seas. Fish have thus been a focus in endocrine disruption studies, and some of the most comprehensive studies on the disruption of sexual development and function are on the roach (Rutilus rutilus). This paper provides a critical analysis of the roach as a sentinel for studies into endocrine disruption. The paper starts by describing what is known on the basic reproductive biology of the roach, information essential for interpreting chemical effect measures on sexual development and function. We then analyze where and how the roach has been applied to improve our understanding of the estrogenic nature of discharges from wastewater treatment works (WWTWs) and describe the phenomenon of feminized male roach in UK rivers. In this paper, the causation of these effects and issues of relative susceptibility and sensitivity of the roach to the effects of estrogenic EDCs are addressed. The paper then describes the ongoing work on the development of genetic and genomic resources for roach and analyses how these are being applied in studies to understand the mechanisms of disruption of sexual development. Finally, the paper addresses the biological significance of sexual disruption and intersex for the individual and discusses the possible implications for wild populations.