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