Environmental transcriptomics under heat stress: Can environmental RNA reveal changes in gene expression of aquatic organisms?

Abstract

To safeguard biodiversity in a changing climate, we require taxonomic information about species turnover and insights into the health of organisms. Environmental DNA approaches are increasingly used for species identification, but cannot provide functional insights. Transcriptomic methods reveal the physiological states of macroorganisms, but are currently species specific and require tissue sampling or animal sacrifice, making community-wide assessments challenging. Here, we test if broad functional information (activity level of the transcribed genes) can be harnessed from environmental RNA (eRNA), which includes extra-organismal RNA from macroorganisms along with whole microorganisms. We exposed Daphnia pulex as well as phytoplankton prey and microorganism colonizers to control (20 °C) and heat stress (28 °C) conditions for seven days. We sequenced eRNA from tank water (after complete removal of Daphnia) as well as RNA from Daphnia tissue, enabling comparisons of extra-organismal and organismal RNA based gene expression profiles. Both RNA types detected similar gene expression responses. Using eRNA, we identified 42 Daphnia genes to be differentially expressed following heat stress. Of these, 62% were also differentially expressed and exhibited similar levels of relative expression in organismal RNA. Both RNA types recovered similar Daphnia heat stress responses via gene ontology terms. In addition to the extra-organismal Daphnia response, eRNA detected community-wide heat stress responses consisting of 199 differentially expressed genes across 9 taxa, and distinct functional profiles via KEGG orthologs. Our study demonstrates that environmental transcriptomics based on eRNA can non-invasively reveal gene expression responses of macroorganisms following environmental changes, with broad potential implications for the biomonitoring of ecological health across the trophic chain.

Environmental RNA as a non-invasive tool for assessing toxic effects in fish: a proof-of-concept study using Japanese medaka exposed to pyrene

“…Of the sequenced reads, more than 92% of the fish tissue RNA were mapped to the reference genome, while 0.6 ± 0.4 % of the eRNA were mapped. This low mapping ratio for eRNA is comparable to 0.5% reported for eRNA of D. pulex (Hechler et al 2022) and indicates that most of the sequenced eRNA originated from non-target microorganisms, such as bacteria (e.g., Aeromonas, Shewanella) and fungi (Figure S7). RNA degradation: RNA-seq analysis usually assumes that the amount of sequenced reads is proportional to the abundance and length of the transcript (Wang et al 2016); however, this assumption can be distorted if RNA degrades.…”

“…eRNA represents a promising avenue for researchers in research fields of ecology and ecotoxicology in this regard. As demonstrated by this study and recent papers (Tsuri et al 2020;Hechler et al 2022), eRNA contains genes involved in a wide range of biological processes and molecular functions and it was able to detect differential gene expressions in response to environmental stressors. While these findings are novel in ecology and ecotoxicology, they are not surprising given that a variety of RNAs have been detected in human sweat and urine (Merchant et al 2017;Bart et al 2021).…”

“…Recent studies reported that the degradation of eRNA for some genes was slower than previously expected (Wood et al 2020;Marshall et al 2021;Jo et al 2022;Kagzi et al 2022). eRNA has been detected for a wide range of aquatic species, including fish (Tsuri et al 2020;Miyata et al 2021;Jo et al 2022), water fleas (Hechler et al 2022;Kagzi et al 2022), polychaetas (von Ammon et al 2019Wood et al 2020), mussels (Marshall et al 2021), ascidians (Wood et al 2020), and algae (Miyata et al 2022). Also, eRNA has been detected for both mitochondrial and nuclear genes (Tsuri et al 2020;Marshall et al 2021;Jo et al 2022) and for several microRNAs (Ikert et al 2021).…”

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

“…Unlike DNA, RNA is only produced for genes that are actively being transcribed, making it a useful tool for understanding the “real time” status of the organisms. Recent studies reported that the degradation of eRNA for some genes was slower than previously expected. − eRNA has been detected for a wide range of aquatic species, including fish, ,, water fleas, , polychaetas, ,, mussels, ascidians, algae, and others . Also, eRNA has been detected for both mitochondrial and nuclear genes ,, and for several microRNAs .…”

“…The eRNA profiles were then compared with transcriptome profiles of whole fish tissue. Comprehensive sequencing of eRNA has previously been reported only for the water flea Daphnia pulex and rainbow trout Oncorhynchus mykiss, making this study potentially valuable in understanding eRNA.…”