Developing an understanding of behavioral and transcriptional implications for the chiral cyanotoxin anatoxin-a and caffeine in common larval fish models.


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Neuroactive compounds are widely and routinely detected aquatic contaminants. With multitudes of chemicals having known and unknown neuroactivity and the sensitivity of early neurodevelopment to contaminant insults, early life stage assays are crucial for understanding hazards of these chemicals to public health and the environment. However, one of the most widely used developmental in vivo assays, the OECD fish embryo toxicity test with zebrafish (Danio rerio), an increasingly common alternative vertebrate model, has been shown to be insensitive to many neuroactive chemicals. Therefore, complimentary approaches such as behavioral and gene expression assays have been used to increase sensitivity, but robust and standardized methods for these endpoints are uncommon. This dissertation primarily focuses on the chiral cyanobacterial neurotoxin anatoxin-a, and considers the neurostimulant caffeine, aiming to elucidate behavioral and transcriptional effects while heeding efficiency of compound and animal use. It further considers environmental relevance, stereospecific effects, species sensitivity, and variability of behavioral assays based on arena size. Specifically, a meta-analysis of the current state of anatoxins research in aquatic systems was performed using environmental exposure distributions (EEDs), and collating bioaccumulation and toxicity data, for which quality was inconsistent. Applying EED information, two alternative vertebrate models, zebrafish and fathead minnows, were exposed to environmentally relevant and higher levels of the commonly studied, synthetic (±) anatoxin-a to compare photolocomotor and gene expression responses, with caffeine as a positive methodological control. Whereas zebrafish were highly insensitive to the racemate, the toxin caused significant hypoactivity and transcriptional changes in fathead minnows. This design was repeated with the naturally produced (+) enantiomer. Mortality was caused by multiple treatment levels in fathead minnows and zebrafish, along with behavioral and gene expression changes, with fathead minnows again being more sensitive to several response variables. To further examine comparative behavioral methods used here and previously, the influence of arena size on behavioral responses of naïve and caffeine exposed zebrafish was investigated to understand inherent variability of the experimental observation arena. This dissertation identified novel anatoxin-a consequences, how effects may be underestimated by studies employing racemic mixtures, and the importance of behavioral methodologies in comparative studies with common fish models.