Mechanistic Understanding of Neurobehavioral Effects of Insecticides on the Larvae of Zebrafish (Danio rerio)

Due to conservation of neuronal functioning across phyla, molecular targets of insecticides are similar in insects and vertebrates. Insecticides thus pose a risk to aquatic vertebrates,such as fish, and potentially cause neurotoxic effects. Although these effects are diverse, neurotoxicity mostly manifests as an alteration in the organism's behavior. Over the lastyears, behavioral endpoints have been used to assess the neurotoxicity of chemicals. A variety of methods, which are able to measure behavioral output from basic locomotion to complexbehaviors, have been developed using fish as a model. Despite the increasing number of studies reporting on chemically induced behavioral effects, knowledge gaps exist regardingthe underlying mechanisms. However, mechanistic knowledge is crucial to truly understand the causes of neurobehavioral effects.This thesis combines ecotoxicological approaches to measure behavior and neurobiological techniques to assess neuronal and neuromuscular effects using larval zebrafish, to investigatethe mechanisms driving behavioral effects in fish. The research focused on eight environmentally relevant insecticides, representing important insecticide classes and modes of action,including acetylcholinesterase inhibitors (diazinon, dimethoate, methomyl, pirimicarb), nicotinic acetylcholine receptor agonists (imidacloprid, thiacloprid), a cation channel modulator(pymetrozine), and an unknown target (flonicamid). Two exposure scenarios were applied: the first allowed to assess short-term effects on spatial distribution of larvae induced by peakexposure; the second focused on locomotor defects induced in early development. Short-term exposure to diazinon and imidacloprid induced aversive responses, which werelinked to olfaction as the response was abolished in olfaction-deficient fish. Neuronal activity mapping indicated that significant changes in brain regions correlated with aversion andstress, suggesting that fish perceiving insecticides as stressful, similar to a change in water chemistry. Developmental exposure induced locomotor defects in all tested concentrations ofcholinergic insecticides. Only exposure to methomyl, imidacloprid, and thiacloprid induced structural changes in the neuromuscular synapse, such as motor axon branching defects or abreak in muscle integrity. Functional and structural defects recovered rapidly for all insecticides except pirimicarb within three days of depuration. Further, there was no particularlysensitive window of development with respect to the behavioral phenotype. Targeted gene expression analysis indicated upregulation of ache (acetylcholinesterase) and smyd1b, a geneinvolved in muscle fiber organization. Regulation of ache and smyd1b may be linked to aberrant axon branching and decreased muscle integrity, respectively.To conclude, the thesis shows that short-term and developmental exposure to insecticides adversely affect fish behavior. Particularly older classes of insecticides pose a higher risk to fishthan newer, more insect-specific ones. This thesis provides mechanistic insights into neuronal processing and emergence of avoidance behavior, and the structural and molecularbase of locomotor defects. This work therefore helps to gain a better understanding of the mechanisms of insecticides on fish behavior as a potential driver of population level effects.