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The antidepressant, venlafaxine (VFX), and climate change stressors, such as increased water temperature and decreased dissolved oxygen, are current threats to aquatic environments. This study aimed to determine how microRNAs (miRNAs) and predicted targeted transcripts were altered in livers of zebrafish exposed to these stressors, and livers of their un-exposed F 1 and F 2 offspring. Following a 21 day exposure to multiple stressors (1 μg/L VFX, +5 °C ambient, 50% O 2), then a subsequent 21 day recovery, relative abundances of cyp3a65 , hsp70 , hsp90 , and ppargc1a and miRNAs predicted to target them (miR-142a, miR-16c, miR-181c, and miR-129, respectively) were measured in the liver via quantitative PCR (RT-qPCR). There were significant decreases in miR-142a in the exposed F 0 generation and the exposed F 1 generation. While there were no changes detected in cyp3a65 relative abundance, there was a significant inverse relationship between cyp3a65 and miR-142a. Hsp70 expression significantly increased in the F 1 generation, which persisted to the F 2 generation and the relative abundance of hsp90 significantly increased in all generations. There was a significant reduction in miR-181c in the F 1 generation, but there was no significant relationship between miR-181c and hsp90. Finally, there was a significant decrease in ppargc1a relative abundance in the F 1 generation which was associated with an increase in miR-129. Combined, these results suggest that parental exposure to multiple, environmentally relevant stressors can confer transcriptional and epigenetic responses in the F 1 and F 2 generations, although identifying which stressor is a driving force becomes unclear. Unlabelled Image • Adult zebrafish were exposed to 21 days of +5 °C, hypoxia (50%), and 1 μg/L venlafaxine. • Zebrafish were returned to control conditions and bred to F 1 and F 2 generations. • Inverse relationships were found between miR-142a and cyp3a65 & miR-129 and ppargc1a. • Hsp70 and hsp90 were significantly increased in the F 1 and F 2 generations. • Multi-stressor exposure confers mRNA and microRNA changes in future generations. [ABSTRACT FROM AUTHOR]

The Grand River watershed is the largest in southern Ontario and assimilates thirty wastewater treatment plants (WWTP) with varied degrees of treatment. Many WWTPs are unable to effectively eliminate several contaminants of emerging concern (CECs) from final effluent, leading to measurable concentrations in surface waters. Exposures to CECs have reported impacts on oxidative stress measured through antioxidative enzymes (SOD, CAT, GPX). This study focuses on the effects of WWTP effluent on four Etheostoma (Darter) species endemic to the Grand River, by investigating if increased antioxidative response markers are present in darter brains downstream from the effluent outfall compared to an upstream reference site relative to the Waterloo, ON WWTP across two separate years (Oct 2020 and Oct 2021). This was assessed using transcriptional and enzyme analysis of antioxidant enzymes and an enzyme involved in serotonin synthesis, tryptophan hydroxylase (tph). In fall 2020, significant differences in transcript markers were found between sites and sexes in GSD with SOD and CAT showing increased expression downstream, in JD with both sexes showing increased SOD downstream, and an interactive effect for tph in RBD. Changes in transcripts aligned with enzyme activity where interactive effects with sex-related differences were observed in fish collected fall 2020. In contrast, transcripts measured in fall 2021 were increased upstream compared to downstream species in RBD and GSD. This study additionally displayed yearly, species and sex differences in antioxidant responses. Continued investigation on the impacts of CECs in effluent in non-target species is required to better understand WWTP effluent impacts. [Display omitted] • Variability in antioxidant response in non-target species exposed to effluent • Antioxidant sex-differences are observed in darters following effluent exposure. • Influence of yearly variation on the antioxidant response • Effluent tolerance varies between species indicated by morphological differences. [ABSTRACT FROM AUTHOR]