Early exposure to atrazine in zebrafish: intergenerational effects and animal personality

Abstract

Animal personality (consistent inter-individual differences in behaviour over time and context) is important for an individual’s fitness. Acute exposure studies in adults show that endocrine disrupting chemicals (EDCs) can deteriorate behavioural consistency and directionally alter personality phenotypes, which may decrease fitness. Early life development is sensitive to environmental stressors and exposure to EDCs may permanently alter phenotypes through to adulthood. In addition, the effects of environmental stressors may not be isolated to a single generation - offspring may indirectly be impacted, via non-genetic processes. Epigenetic mechanisms (e.g. DNA methylation) that help to regulate the genome, can become permanently altered via EDC exposure during early life and alterations have the potential to be inherited in the germ cells. The aims of this thesis were to investigate direct effects of the EDC, atrazine, on animal personality in the zebrafish (Danio rerio) and the indirect effects on personality in their offspring. Recent studies suggest that zebrafish inherit the methylome (total DNA-methylation marks across the genome) in an unchanged state from their fathers, suggesting a possible route for environmental specific information to be inherited. Hence, I focus on paternal effects in this thesis. In addition, I investigate the underlying basis of mRNA transcript number differences in F1 zebrafish brain tissue, with the aim to determine whether observed changes in behaviour are underpinned by changes in gene expression. This is the first study to examine the effects of atrazine on personality, for both direct and indirect exposure.

Juvenile zebrafish were exposed to atrazine at typical environmental concentrations: 0.3, 3 or 30 part per billion (ppb) through sexual differentiation and their personality phenotypes were assayed (activity, aggression, boldness, anxiety and exploration) at adulthood. F1 offspring were produced from atrazine exposed and control males, mated with unexposed females, creating full-sibling families. Progeny personality was tested at adulthood. Brain samples of a subset of F1 progeny (based on anxiety and aggression phenotypes) were taken to determine mRNA transcript number of candidate genes involved in the regulation of the serotonergic system (slc6a4a, slc6a4b, htr1Aa, htr1B, htr2B), an underlying component involved in anxiety and aggressive-related behaviours, and a potential target of atrazine.

I found evidence that direct and paternal atrazine exposure significantly influences aspects of boldness, aggression, anxiety and exploration, though effects were not consistent across all doses. Changes in boldness were sex-dependent, specifically, F0 female boldness was reduced in a dose-dependent manner and F0 males exhibited a threshold increase in boldness. F1 females exhibited a similar dose-dependent reduction in boldness but in the opposite direction to F0 females and there was little change amongst F1 males. There was some evidence of reduced aggression after direct exposure, but paternal atrazine exposure significantly reduced aggression in the 0.3ppb and 3ppb groups. No F0 treatment differences in activity were observed, however, significant F1 decreases in activity were observed during aggression testing in the 0.3ppb and 3ppb groups. Anxiety increased and exploration decreased significantly in the F0 0.3ppb group only, while in the F1’s there was some evidence of alterations in anxiety and exploration, but not significantly so.

Paternal atrazine exposure significantly down-regulated htr1Aa mRNA expression in females and slightly up-regulated it in males; moreover, htr1Aa mRNA (and slc6a4a - but not significantly so) was found to correlate positively with anxiety levels in controls; however, this relationship was disrupted in the atrazine treatment group. The candidate genes tested here did not explain the significant reduction in aggression observed in the offspring of atrazine treated fathers, though both slc6a4b and htr1B mRNA correlated negatively with an aggressive personality phenotype.

In conclusion, early developmental exposure to environmental doses of atrazine resulted in persistent changes in personality phenotypes through to adulthood and effects were present in the offspring of atrazine exposed fathers. Moreover, some aspects of the serotonergic system were disrupted in the progeny. Overall, these results add to the ecological consequences of environmental contaminants, most importantly, that effects may be further propagated down the germ line.