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

Abstract

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.

Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

“…We found that expression levels of cat and actb were relatively higher in eRNA compared to in fish whole body, and also found that expression levels of cat in eRNA increased by CPS exposure, whereas those in fish whole body decreased. These findings are consistent with our recent study demonstrating that differentially expressed genes in response to pyrene exposure were not identical between eRNA and fish whole body (Hiki et al., 2023). In addition, a recent study found that DNA methylation patterns were different between eDNA and DNA extracted from the host organism ( L. stagnalis ), possibly because eDNA is predominantly released from dysfunctional cells that are hypermethylated compared to other cells (Zhao et al., 2023).…”

“…We found that expression levels of cat and actb were relatively higher in eRNA compared to in fish whole body, and also found that expression levels of cat in eRNA increased by CPS exposure, whereas those in fish whole body decreased. These findings are consistent with our recent study demonstrating that differentially expressed genes in response to pyrene exposure were not identical between eRNA and fish whole body (Hiki et al, 2023).…”

“…Oryzias latipes was selected as the model species due to its widespread use in standardized fish toxicity tests, as recommended by the OECD guideline (OECD, 2019). The details of stock maintenance were described elsewhere (Hiki et al, 2023). Briefly, brood stock was kept in 5 L glass tanks with dechlorinated tap water (hardness: about 80 CaCO 3 mg/L) at 25°C under 16-8 h light-dark cycles.…”

“…As eRNA is less stable than eDNA (Jo et al., 2022; Kagzi et al., 2022), eRNA can reduce false positives associated with detecting dead organisms, thereby providing a real‐time ‘snapshot’ of a living species (Giroux et al., 2022; Littlefair et al., 2022; Miyata et al., 2021). Furthermore, a variety of genes have been detected in eRNA (Tsuri et al., 2020), including stress‐ and defense‐related genes (Hechler et al., 2023; Hiki et al., 2023). The application of high throughput RNA sequencing (RNA‐Seq) to laboratory experiments revealed that the expression profiles in eRNA were altered in response to environmental stress such as exposure to pyrene (Hiki et al., 2023) and increased temperature (Hechler et al., 2023).…”

“…To develop a method for the relative quantification of eRNA for a single species, we performed toxicity tests using Japanese medaka Oryzias latipes and conducted relative gene expression analysis of catalase ( cat ), an antioxidant gene, in eRNA collected from surrounding water, using qPCR. The selection of cat was based on its detection in eRNA in our previous study (Hiki et al., 2023). We selected two chemicals with different toxic modes of action: an organophosphate insecticide chlorpyrifos (CPS) and an anticonvulsant carbamazepine (CBZ).…”