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