Environmental RNA as a Noninvasive Tool for Assessing Toxic Effects in Fish: A Proof-of-concept Study Using Japanese Medaka Exposed to Pyrene

Although environmental RNA (eRNA) is emerging as a noninvasive
tool to assess the health status of aquatic macroorganisms, the potential
of eRNA in assessing chemical hazards remain largely untested. In
this study, we investigated the ability of eRNA to detect changes
in gene expression in Japanese medaka fish (Oryzias
latipes) in response to sublethal pyrene exposure,
as a model toxic chemical. We performed standardized acute toxicity
tests and collected eRNA from tank water and RNA from fish tissue
after 96 h of exposure. Our results showed that over 1000 genes were
detected in eRNA and the sequenced read counts of these genes correlated
with those in fish tissue (r = 0.50). Moreover, eRNA
detected 86 differentially expressed genes in response to pyrene,
some of which were shared by fish RNA, including the suppression of
collagen fiber genes. These results suggest that eRNA has the potential
to detect changes in gene expression in fish in response to environmental
stressors without the need for sacrificing or causing pain to fish.
However, we also found that the majority of sequenced reads of eRNA
(>99%) were not mapped to the reference medaka genome and they
originated
from bacteria and fungi, resulting in low sequencing depth. In addition,
eRNA, in particular nuclear genes, was highly degraded with a median
transcript integrity number (TIN) of <20. These limitations highlight
the need for future studies to improve the analytical methods of eRNA
application.