Effect of paternal exposure to glyphosate and microcystin-LR on zebrafish (Danio rerio) fathers and progeny: An integrated investigation based on behavioral, histopathological, biochemical, and transcriptomic analysis

The coexistence of glyphosate (GLY) and microcystin-LR (MC-LR) in the environment has become a problem that cannot be ignored. However, the combined toxic effects of these compounds on aquatic organisms remain underexplored. The purpose of this study was to evaluate the individual and combined effects of GLY and MC-LR on the neurobehavior of exposed adult zebrafish and their untreated offspring (F1 larvae), as well as to explore the underlying mechanisms. In this study, six month old male zebrafish were exposed to 3.5 mg/L GLY and 35 μg/L MC-LR, alone and in combination, for 21 d. The results revealed that exposure to GLY and/or MC-LR decreased locomotor activity, heightened anxiety-like behaviors, and disrupted social interactions in adult zebrafish, thereby confirming their neurotoxic effects. Pathological damage, along with elevated levels of ROS, IL-1β, and caspase-3, were observed in the brains of the treated fish. RNA-seq analysis further revealed that GLY and/or MC-LR treatment induced widespread transcriptional changes in the zebrafish brain, potentially impacting a range of biological functions, including neurobehavior. Meanwhile, paternal exposure to GLY and MC-LR, either individually or in combination, inhibited the spontaneous movement of untreated embryos at 24 hpf and reduced swimming activity at 120 hpf. Transcriptome sequencing of larvae further elucidates the intricate mechanisms underlying the intergenerational neurotoxicity induced by GLY and/or MC-LR, and the metabolic alterations potentially play an important role in mediating this toxicity. Overall, these findings collectively provide robust evidence of intergenerational effects resulting from exposure to individual and combined GLY and MC-LR, especially regarding persistent impacts on behavioral parameters, which should be emphasized in future ecological and health risk assessments.