# Pattern of Detections Across Multiple Environmental Messenger RNAs (e‐mRNAs) in Stressor‐Exposed Zebrafish (Danio rerio)

## TL;DR

This study explores how environmental messenger RNAs (e-mRNAs) can detect stress in zebrafish exposed to a chemical pollutant, showing they decay quickly and indicate recent organism presence.

## Contribution

The study introduces e-mRNAs as a potential tool for monitoring environmental stressor impacts on aquatic organisms.

## Key findings

- e-mRNA detection rates dropped significantly beyond 3 hours after fish removal, with most undetectable by 72 hours.
- PFOS exposure did not cause observable shifts in e-mRNA concentrations, possibly due to tissue-specific gene responses.
- e-mRNAs could indicate recent organism presence and may detect contaminant impacts under the right conditions.

## Abstract

Environmental RNA (eRNA) is gaining ground as an environmental monitoring tool. Whereas eDNA is mainly utilized for species detection, eRNA may provide additional classes of inference. The comparatively more rapid signal decay rates of eRNA provide narrower temporal windows for species presence, while detection of environmental messenger RNAs (e‐mRNAs) could provide evidence of genomic responses to environmental stressors. We explored e‐mRNA as an environmental tracer for stress imposed on animal populations by investigating the decay dynamics of e‐mRNA gene detections from target organism presence to recent presence. We tested seven select e‐mRNAs of known molecular targets of perfluorooctanesulfonic acid (PFOS) toxicity in tanks containing zebrafish (
Danio rerio
) exposed to an environmentally relevant concentration of PFOS. eRNA samples were collected just prior to fish removal following a 21‐day exposure and continued over nine timepoints across 3 days. The quantity and quality of total eRNA declined over time for both treatments, but were still detectable at 72 h post fish removal. The PFOS exposure failed to elicit observable shifts in e‐mRNA target concentrations compared to control tanks, perhaps because the selected gene targets are primarily responsive to PFOS in liver and kidney, which may not contribute strong eRNA signatures. Detection rates for all e‐mRNAs dropped significantly beyond 3 h post fish removal, with most being undetectable by 72 h. The signal lifespan of e‐mRNAs in this study implies that the detection of such traces will be a strong indicator of target organism presence (or recent presence), and that given the right combination of stressor concentrations, impacted tissues or organs, and gene targets, contaminant impacts on organism health should be detectable in environmental samples. Future studies targeting toxicologically effective stressor doses for well‐established gene targets will be an important advancement in establishing the utility of e‐mRNA as a noninvasive environmental stressor monitoring tool.

We evaluated e‐mRNAs of known molecular targets of perfluorooctanesulfonic acid (PFOS) toxicity in zebrafish in a controlled laboratory experiment. The detection patterns of e‐mRNAs in our study imply that diminishing detection of these traces over time is a strong indicator of target organism presence (or very recent presence), providing a narrower timeframe of detection than eDNA provides. Our study also suggests that contaminant impacts on organism health should be detectable in water samples, given the right combination of stressor concentrations, impacted tissues or organs, and gene targets.