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.

Recent developments in environmental DNA (eDNA) analysis allow more rapid and extensive biomonitoring than traditional capture‐based surveys do. However, detection of eDNA not derived from living organisms may lead to false‐positive inferences of species presence. Such limitations may be overcome by utilizing RNA molecules present in the environment (environmental RNA [eRNA]) because of their physiochemical instability. Nevertheless, the biomonitoring performance of eRNA analysis remains unclarified because of the substantial lack of knowledge regarding basic eRNA properties, such as its persistence and degradation mechanisms. Here, we performed a factorial aquarium experiment to assess the effects of water temperature (10, 20, and 30°C) and pH (4, 7, and 10) conditions on the degradation of zebrafish (Danio rerio) eDNA and eRNA, targeting the mitochondrial cytochrome b (CytB) and nuclear beta‐2‐microglobulin (b2m) genes. A linear mixed‐model analysis showed that the degradation of eRNA was significantly faster than that of eDNA. Higher water temperatures promoted both eDNA and eRNA degradation, and alkaline conditions substantially promoted eRNA but not eDNA degradation. This might be explained by the physicochemical characteristics of DNA and RNA molecules, the membranous structure surrounding them, and their susceptibility to environmental microbial activity. Moreover, the relative concentrations of zebrafish eRNA to eDNA decreased over time, inferring that the ratio of eRNA to eDNA concentrations can be used for estimating the elapsed time since the genomic material was released and the freshness of the target eDNA signal in the field. Nevertheless, given that the confidence intervals of the eDNA and eRNA decay rates tended to overlap for each treatment level, this study indicates that fish eRNA is not always degraded rapidly and is, in fact, more abundant in water than previously expected. This result favors the application of eRNA analysis to indicate living biotic assemblages.

Environmental DNA (eDNA) analysis has enabled more sensitive and efficient biological monitoring than traditional methods. However, since the target species is not directly observed, interpretation of results cannot preclude process Type I errors. Specifically, there may be a spatial or temporal gap between the target eDNA and the eDNA source in the sampled area. Moreover, eDNA surveillance lacks the ability to distinguish whether eDNA originated from a living or non-living source. This kind of Type I error is difficult to control for, in part, because the relationship between the state of eDNA (i.e., intracellular or extracellular) and the degradation rate is still unclear. Here, we applied PMA (Propidium monoazide) to eDNA analysis which enabled us to differentiate “intact cells” from “disrupted cells.” PMA is a dye that has a high affinity for double-stranded DNA and forms a covalent bond with double-stranded DNA and inhibits amplification of the bonded DNA molecules by PCR. Since PMA is impermeable to the cell membrane, DNA protected by an intact cell membrane can be selectively detected. In this study, we investigated the workability of PMA on vertebrate eDNA using zebrafish, Danio rerio. Aquarium water was incubated for 1 week to monitor the eDNA degradation process of both intracellular and extracellular eDNA. We developed ten species-specific quantitative PCR assays for D. rerio with different amplification lengths that enabled independent quantification of total eDNA (sum of intracellular and extracellular eDNA, commonly measured in other studies) and intracellular eDNA (DNA in intact cells) and allow for analyses of sequence length-dependent eDNA degradation in combination with PMA. We confirmed that PMA is effective at differentiating “intact” and “disrupted” fish cells. We found that total eDNA and intracellular eDNA have different degradation processes that are dependent on the length of target sequence. For future conservation efforts using eDNA analyses, it is necessary to increase the reliability of the analysis results. The research presented here provides new analysis tools that expand our understanding of the ecology of eDNA, so that more accurate and reliable conclusions can be determined.

Environmental RNA (eRNA) analysis has recently received attention as a better means to infer the physiological status of a community and living biotic assemblages than environmental DNA (eDNA). However, eRNA is thought to be degraded more rapidly than eDNA, increasing the risk of false-negative detection and complicating large-scale eRNA sampling in the field. In addition, the need for a deep freezer (− 80°C or below) further limits the practical application of eRNA analysis in places that are not accessible by vehicle. Here we focused on two types of reagents (RNAlater and LBP buffer) and assessed their performance for eRNA preservation. We found that very high concentrations of ayu (Plecoglossus altivelis) eRNA were quantifiable from filter samples collected from an aquarium by using RNAlater preservation at − 20°C for at least a week. Compared with the sample stored at − 20°C without any preservative, the filter samples preserved in RNAlater had eRNA concentrations that were tens to hundreds of times higher. Although further technical refinement is needed, our findings have provided valuable information to enhance the methodology for improving eRNA quality and quantity in environmental samples. This will boost the practical application of eRNA-based meta-transcriptomics targeting macro-organisms.

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