Messenger RNA typing of environmental RNA (eRNA): A case study on zebrafish tank water with perspectives for the future development of eRNA analysis on aquatic vertebrates

Abstract

As an alternative/supplement to conventional biodiversity survey methods, environmental DNA (eDNA) analysis has developed rapidly during the past decade, and is widely used for the biomonitoring of wildlife. However, the interpretation of eDNA results has been limited to the presence/absence or biomass/abundance of the target species because of uncertainty regarding the dynamics of eDNA in natural environments. This limitation may be ameliorated by targeting environmental RNA (eRNA). RNA is more prone to degrade than DNA and the pattern of messenger RNA (mRNA) expression changes depending on physiological conditions, meaning that the presence or concentration of mRNA could reflect the organism's presence with higher temporal resolution and provide information beyond simple presence/absence. Technical developments in the detection of eRNA focusing on mRNA with these distinct features could permit the advanced usage of genetic materials in water. In advancing this technique, we initiated this study asking that if we can detect elevated levels of eRNA whose genes are specific to a tissue source (e.g., gills or skin), then could not we infer the tissue origin of the genetic material detected. To this end, we developed gene‐specific primer sets for the target genes with biased expression in the gills, skin, and intestine, and conducted reverse transcription–polymerase chain reaction on zebrafish breeding tank water samples, obtaining positive results for all assays. The result of our experiment confirmed that the specific target tissues can be the source of genetic materials detected in water and with that we offer a proof of concept for eRNA analysis targeting specific mRNAs of aquatic vertebrates. In this commentary, we provide information on the experimental steps used for mRNA typing of eRNA from zebrafish as well as the limitations and challenges of this technique and the prospect of mRNA typing of eRNA in the future.

Reconstruction of 100-year dynamics in Daphnia spawning activity revealed by sedimentary DNA

“…For Daphnia , the mRNA-cDNA of the Chk1 gene regulates oocyte maturation during reproduction 53 and Caspase-3 mRNA increases with aging related to the appearance of sexual reproduction 54 . Recent studies have indicated advances in these techniques by demonstrating that eRNA can be detected from water 55 and sediment core samples 56 . Furthermore, it has been shown by using sedimentary mRNA that some zooplankton were surviving in dormancy in sediments and possibly use cytochrome c oxidase (COX) to exit dormancy 56 .…”

Fine-tuning the performance of abundance estimation based on environmental DNA (eDNA) focusing on eDNA particle size and marker length

“…To explain the mechanism underlying the findings in this study, future empirical research on the production source and persistence state of eDNA is required. For instance, detecting tissue-specific messenger RNA in water could identify the production source of eDNA (Tsuri et al, 2021). Alternatively, visualization of eDNA particles via fluorescence in situ hybridization (FISH) techniques and immunostaining (e.g., Kogure et al, 2006;Amann & Fuchs, 2008) may directly offer information on its cellular and the molecular structure.…”

Plant-animal interactions in the era of environmental DNA (eDNA) – a review

“…Recent methodological developments to collect and extract environmental RNA (eRNA) might also be leveraged to understand changes of gene expression with physical and biological pressure (functional genomics; Tsuri et al, 2021), with possibilities of expansion into ecological epigenetics, ecosystem health, func-tional metagenomics, population-level inference, or even the interface of species-species interactions (e.g., Stewart & Taylor, 2020;Veilleux et al, 2021). Unlike eDNA, eRNA can go beyond species and PAI quantification, such as understanding life histories, ages, metabolic activities, physiological conditions and health of interacting organisms.…”