Profiling microRNA expression in Atlantic killifish (Fundulus heteroclitus) gill and responses to arsenic and hyperosmotic stress

•RNA-seq analysis identified 270 microRNAs expressed in Atlantic killifish gill.•miR-135b was induced by salinity and predicted to target osmoregulatory genes.•Arsenic exposure disrupted miR-135b expression alone and in response to salinity. The Atlantic killifish (Fundulus heteroclitus), native to estuarine areas of the Atlantic coast of the United States, has become a valuable ecotoxicological model as a result of its ability to acclimate to rapid environmental changes and adapt to polluted habitats. MicroRNAs (miRNAs) are highly conserved small RNAs that regulate gene expression and play critical roles in stress responses in a variety of organisms. Global miRNA expression in killifish and the potential roles miRNA have in environmental acclimation have yet to be characterized. Accordingly, we profiled miRNA expression in killifish gill for the first time and identified a small group of highly expressed, well-conserved miRNAs as well as 16 novel miRNAs not yet identified in other organisms. Killifish respond to large fluctuations in salinity with rapid changes in gene expression and protein trafficking to maintain osmotic balance, followed by a secondary phase of gene and protein expression changes that enable remodeling of the gills. Arsenic, a major environmental toxicant, was previously shown to inhibit gene expression responses in killifish gill, as well the ability of killifish to acclimate to a rapid increase in salinity. Thus, we examined the individual and combined effects of salinity and arsenic on miRNA expression in killifish gill. Using small RNA sequencing, we identified 270 miRNAs expressed in killifish, and found that miR-135b was differentially expressed in response to arsenic and at 24 h following transfer to salt water. Predicted targets of miR-135b are involved in ion transport, cell motility and migration, GTPase mediated signal transduction and organelle assembly. Consistent with previous studies of these two environmental stressors, we found a significant interaction (i.e., arsenic dependent salinity effect), whereby killifish exposed to arsenic exhibited an opposite response in miR-135b expression at 24 h post hyperosmotic challenge compared to controls. By examining mRNA expression of predicted miRNA targets during salinity acclimation and arsenic exposure, we found that miR-135b targets were significantly more likely to decrease during salinity acclimation than non-targets. Our identification of a significant interaction effect o