Warm temperature and alkaline conditions accelerate environmental RNA degradation

Abstract

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.

Simple and efficient preservation of fish environmental RNA in filtered water samples via RNAlater

“…Throughout water sampling and ltration, disposable gloves and masks were worn to prevent RNase contamination in the water samples. All equipment for water ltration ( lter funnels and tweezers) was bleached before use in 0.1% sodium hypochlorite solution for at least 5 min (Yamanaka et al, 2017;Jo et al, 2022).…”

“…Then, using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real-Time; Takara Bio Inc., Japan), gDNA was enzymatically removed from the eRNA extracts (in total, two rounds of gDNA removal) and complementary DNA (cDNA) was immediately synthesized (Jo et al, 2022). Following the manufacturer's instructions, 20 µL of cDNA sample was prepared from 7 µL of each eRNA extract, for which random and oligo dT primers were used for reverse transcription.…”

“…The rapid degradation of eRNA may occur during the transportation of samples from the eld to the laboratory, which in ates the risk of false-negative eRNA detection, underestimates eRNA concentration in a sample, and complicates eRNA sampling at multiple sites or at high frequencies in the eld. Previous studies have reported the detection/quanti cation of eRNA released by sh and macroinvertebrates in laboratory conditions (e.g., aquarium experiments), in which lter samples were immediately transferred to RNA extraction (Tsuri et al, 2021;Jo et al, 2022) or stored at − 80°C until extraction (Wood et al, 2020;Marshall et al, 2021). However, the need for equipment to freeze eRNA samples (− 80°C or below, desirable for the stable preservation of RNA molecules; e.g., a deep freezer) would limit the practical application of eRNA analysis to areas that were accessible only by foot.…”

“…However, it is also believed that eRNA degrades more rapidly and persists for a shorter time in the water than eDNA owing to its physicochemical instability (Marshall et al, 2021;Jo et al, 2022;Kagzi et al, 2022). The rapid degradation of eRNA may occur during the transportation of samples from the eld to the laboratory, which in ates the risk of false-negative eRNA detection, underestimates eRNA concentration in a sample, and complicates eRNA sampling at multiple sites or at high frequencies in the eld.…”

Comparative environmental RNA and DNA metabarcoding analysis of river algae and arthropods for ecological surveys and water quality assessment

“…Qian et al 21 investigated the temperature effects on the stability of F. chinensis eDNA/eRNA and found that eRNA showed a faster degradation if temperature was increased, and the stability was maintained across the range of temperature. Furthermore, Kagzi et al 21 reported eRNA of Daphnia pulex degrades more rapidly than eDNA across a broad range of pH condition and Jo et al 22 reported that warm temperature and alkaline conditions accelerate the degradation rate of Danio rario eRNA. The degradation rate of eRNA would vary according to species, genes, environmental conditions, and analytical method.…”