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Abstract Molecular detection of environmental DNA (eDNA) and RNA (eRNA) allows highly sensitive qualitative (i.e., presence or absence) and quantitative (i.e., abundance) monitoring of aquatic bacteria. However, bacterial molecular diagnostics are limited by low positive predictive values. Protocols for bacterial eDNA and eRNA molecular monitoring have primarily focused on optimizing specimen collection, and the optimal method to purify bacterial nucleic material from postcollection aquatic specimens to maximize the analytical sensitivity of molecular diagnostics remains poorly defined. Accordingly, strategies to isolate bacterial eDNA and eRNA from fresh and saltwater were investigated. We evaluated two filtration and four nucleic acid purification systems as representative of current generation bacterial eDNA and eRNA isolation strategies for capacity to isolate bacterial eDNA and eRNA from prelysed (i.e., free‐nucleic acids) and viable (i.e., colony forming units, CFU) bacterial cells. We also compared the sensitivities of reverse transcription quantitative PCR (RT‐qPCR) and metagenomic shotgun microbiome sequencing. The optimal protocol used 0.7 μm borosilicate glass filters (Whatman plc) followed by extraction with the RNeasy PowerWater kit (Qiagen). The protocol had a very high analytical sensitivity (10−3–100 ng and 102–101 CFU detected in 500 mL) across multiple species of bacteria, when tested with either RTqPCR or metagenomic sequencing. Importantly, this study highlighted several limitations which are restrictive to both qualitative and quantitative bacterial eDNA and eRNA studies. First, a 12‐h time course between sampling and extraction revealed significant species‐specific changes in cell number and free‐nucleic acid concentrations can occur postspecimen collection. Second, we found Gram‐positive bacteria yielded less nucleic material compared to Gram‐negative bacteria suggesting bacterial eDNA and eRNA studies could be biased by microorganism genome stability and extraction efficiency. This study highlights the need to define the species‐specific diagnostic sensitivity of a protocol when monitoring aquatic bacterial eDNA and eRNA with molecular diagnostics.

The analyses of environmental DNA (eDNA) and environmental RNA (eRNA) released by organisms into their surrounding environment (water, soil and air) have emerged as powerful tools for monitoring biodiversity. While eDNA has been widely adopted for the non‐invasive detection of species and characterization of community composition, the utilization of eRNA is still in its infancy. Due to its functional nature, eRNA holds intriguing potential for biodiversity monitoring offering new avenues of research beyond species detection. For example, conspecifics that are almost genetically identical can exhibit distinct transcriptomic differences depending on their life stage. In this issue of Molecular Ecology Resources, Parsley and Goldberg (2024) demonstrate, through a lab‐validated field study, that eRNA can be used to detect distinct life stages of amphibians. This study elegantly demonstrates that eRNA can be used not only to detect invasive or endangered species but also to reveal population demographic information important for guiding effective conservation strategies. [ABSTRACT FROM AUTHOR]

Abstract Environmental RNA (eRNA) has the potential as a non‐invasive tool for assessing the physiological status of macro‐organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration‐response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta‐actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription‐quantitative polymerase reaction (RT‐qPCR) method, and the sensitivity of the ddPCR assay was 10‐fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross‐react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter‐assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT‐qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations. [ABSTRACT FROM AUTHOR]

Nucleic acids released by organisms and isolated from environmental substrates are increasingly being used for molecular biomonitoring. While environmental DNA (eDNA) has received much attention, the potential of environmental RNA as a biomonitoring tool remains under‐explored. Several recent studies using paired DNA and RNA metabarcoding of bulk samples suggest that RNA might better reflect "metabolically active" parts of the community. However, such studies mainly capture organismal eDNA and eRNA. For larger eukaryotes, isolation of extra‐organismal RNA will be important, but viability needs to be examined in a field‐based setting. In this study we evaluate (a) whether extra‐organismal eRNA release from macroeukaryotes can be detected given its supposedly rapid degradation, and (b) if the same field collection methods for eDNA can be applied to eRNA. We collected eDNA and eRNA from water in lakes where fish community composition is well documented, enabling a comparison between the two nucleic acids in two different seasons with monitoring using conventional methods. We found that eRNA is released from macroeukaryotes and can be filtered from water and metabarcoded in a similar manner as eDNA to reliably provide species composition information. eRNA had a small but significantly greater true positive rate than eDNA, indicating that it correctly detects more species known to exist in the lakes. Given relatively small differences between the two molecules in describing fish community composition, we conclude that if eRNA provides significant advantages in terms of lability, it is a strong candidate to add to the suite of molecular monitoring tools. [ABSTRACT FROM AUTHOR]

While maritime transport boosts global trade by shipping bulk goods, it raises concerns about the spread of harmful bacteria via ballast water. Moreover, the dark and cold environments of ballast tanks often harbor extracellular DNA from dead organisms, leading to false positives in traditional environmental DNA (eDNA) metabarcoding analyses. Here, we alternatively employed environmental RNA (eRNA) metabarcoding to assess its potential for reducing false positive in ballast water monitoring. We collected eDNA and eRNA in parallel from ballast water before and after disinfection in three vessels. High-throughput sequencing of the 16S rRNA V4-V5 regions and cDNA counterparts was conducted to compare bacterial community composition. Our findings showed that over 80 % of the top 150 abundant amplicon sequence variants (ASVs) were detected by both eRNA and eDNA metabarcoding. Samples sequenced separately using DNA and RNA consistently clustered together, indicating similar community recovery efficacy. However, 42 % of ASVs were detected exclusively in DNA, resulting in significantly higher bacterial diversity compared to RNA, which suggests false positives in the DNA method. In treated samples with higher dead cell counts, the RNA method showed significantly lower bacterial diversity, indicating its effectiveness in detecting live bacteria. In summary, eRNA metabarcoding offers comparable recovery efficiency while maintaining a lower false-positive rate than eDNA metabarcoding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lake sediments accumulate information on biological communities thus acting as natural archives. Traditionally paleolimnology has focussed on fossilized remains of organisms, however, many organisms do not leave fossil evidence, meaning major ecosystem components are missing from environmental reconstructions. Many paleolimnology studies now incorporate molecular methods, including investigating microbial communities using environmental DNA (eDNA), but there is uncertainty about the contribution of living organisms to molecular inventories. In the present study, we obtained DNA and RNA inventories from sediment spanning 700 years to investigate the contribution of past and active communities to the molecular signal from sedimentary archives. Additionally, a droplet digital PCR (ddPCR) targeting the 16S ribosomal RNA (16S rRNA) gene of the photosynthetic cyanobacterial genera Microcystis was used to explore if RNA signals were from legacy RNA. We posit that the RNA signal is a mixture of legacy RNA, dormant cells, living bacteria and modern‐day trace level contaminants that were introduced during sampling and preferentially amplified. The presence of legacy RNA was confirmed by the detection of Microcystis in sediments aged to ~200 years ago. Recent comparisons between 16S rRNA gene metabarcoding and traditional paleo proxies showed that past changes in bacterial communities can be reconstructed from sedimentary archives. The recovery of RNA in the present study has provided new insights into the origin of these signals. However, caution is required during analysis and interpretation of 16S rRNA gene metabarcoding data especially in recent sediments were there are potentially active bacteria. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) technology has been successfully applied to detect organisms in various aquatic ecosystems. However, eDNA has been proven to exist for a long time in environmental samples. The timeliness of eDNA detection results largely depends on the rate of molecular degradation. Environmental RNA (eRNA) is considered an excellent complementary tool because most researchers believe that RNA degrades faster than DNA in vitro, while, to the best of our knowledge, the number of published articles related to eRNA is very limited. To address an important knowledge gap, this study focused on the response mechanism of eRNA degradation to water temperature change as compared with eDNA. Changes in the concentration of eDNA and eRNA of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from Fenneropenaeuschinensis were detected at four temperatures (10, 15, 20 and 25 °C). The results showed that the degradation rate of eDNA increased with an increase in temperature. The degradation rate constants ranged from 0.011 to 0.486 h−1 and the degradation time ranged from 8 to 383 h for eDNA. The degradation rate of eRNA changed slightly with an increase in temperature. The degradation rate constants ranged from 0.190 to 0.379 h−1 and the degradation time ranged from 11 to 22 h for eRNA. eRNA showed better stability under temperature change and maintained a faster degradation rate at low temperatures. These results provide answers to the questions of whether eRNA and eDNA degradation rates are fast or slow. Furthermore, this study may suggest the potential superiority of eRNA over eDNA and promote further study of eRNA in future research. [ABSTRACT FROM AUTHOR]

Background: Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems. Methods: Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV. Results: The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed. [ABSTRACT FROM AUTHOR]

Metabarcoding analysis using environmental DNA (eDNA) is increasingly being used for monitoring biota. However, knowledge regarding abundance of fish environmental RNA (eRNA) and its utility in ecological survey is limited. In this study, we aimed to perform eDNA/eRNA metabarcoding analyses of fishes using water samples from rivers in Japan. Sufficient amounts of eRNA from various species were present in the environmental water samples, which can support ecological survey based on eRNA metabarcoding. The sensitivity of eRNA was equivalent to that of eDNA; however, its positive predictivity was superior to that of eDNA. In addition, correlation with abundance in the ecological survey was higher for eRNA than for eDNA. Only eRNA (and the ratio of eRNA and eDNA) enabled distinguishing saltwater or brackish water fishes (false positives) that could be generated due to contamination from domestic wastewater containing their corpses or body parts or from bird feces. To the best of our knowledge, this is the first report of the possibility of eRNA to serve as a critical indicator of living biotic assemblages that actually inhabit sampling points.

Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. While DNA sequencing captures both alive and dead organisms, environmental RNA (eRNA) better targets living organisms or organisms of recent origin in the environment. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in (i) biodiversity of multiple microeukaryotic communities; and (ii) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2%–6.9%), when compared to low OC sediments (1.2%–2.8%). Dissimilarity in community composition increased for all three groups between Low OC and High OC, as well as the classified feeding type of nematode genera (with more nonselective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon‐like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of benthic microeukaryotes could be a powerful tool in recurring monitoring programmes. [ABSTRACT FROM AUTHOR]

Current advancements in environmental RNA (eRNA) exploit its relatively fast turnover rate relative to environmental DNA (eDNA) to assess 'metabolically active' or temporally/spatially recent community diversity. However, this focus significantly underutilizes the trove of potential ecological information encrypted in eRNA. Here, we argue for pushing beyond current species-level eDNA detection capabilities by using eRNA to detect any organisms with unique eRNA profiles, potentially including different life-history stages, sexes, or even specific phenotypes within a species. We also discuss the future of eRNA as a means of assessing the physiological status of organisms and the ecological health of populations and communities, reflecting ecosystem-level conditions. We posit that eRNA has the potential to significantly improve the resolution of organism detection, biological monitoring, and biomonitoring applications in ecology. eDNA has revolutionized species-detection and biomonitoring applications. Recent research has shown that eRNA can be extracted from aquatic environments. Current eRNA applications exploit its rapid degradation to quantify temporally recent or metabolically active communities. Next-generation sequencing (NGS) provides unprecedented ability to quantify the transcriptome. The application of NGS to study eRNA could significantly improve the resolution of organism detection and biological monitoring. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) technology has been successfully applied to detect organisms in various aquatic ecosystems. However, eDNA has been proven to exist for a long time in environmental samples. The timeliness of eDNA detection results largely depends on the rate of molecular degradation. Environmental RNA (eRNA) is considered an excellent complementary tool because most researchers believe that RNA degrades faster than DNA in vitro, while, to the best of our knowledge, the number of published articles related to eRNA is very limited. To address an important knowledge gap, this study focused on the response mechanism of eRNA degradation to water temperature change as compared with eDNA. Changes in the concentration of eDNA and eRNA of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from Fenneropenaeuschinensis were detected at four temperatures (10, 15, 20 and 25 °C). The results showed that the degradation rate of eDNA increased with an increase in temperature. The degradation rate constants ranged from 0.011 to 0.486 h−1 and the degradation time ranged from 8 to 383 h for eDNA. The degradation rate of eRNA changed slightly with an increase in temperature. The degradation rate constants ranged from 0.190 to 0.379 h−1 and the degradation time ranged from 11 to 22 h for eRNA. eRNA showed better stability under temperature change and maintained a faster degradation rate at low temperatures. These results provide answers to the questions of whether eRNA and eDNA degradation rates are fast or slow. Furthermore, this study may suggest the potential superiority of eRNA over eDNA and promote further study of eRNA in future research.

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid-liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2. •Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Ribosomal RNA (rRNA) is used widely to investigate potentially active microorganisms in environmental samples, including soil microorganisms and other microbial communities that are subjected to pronounced seasonal variation in temperature. This raises a question about the turnover of intracellular microbial rRNA at environmentally relevant temperatures. We analyzed the turnover at four temperatures of RNA isolated from soil bacteria amended with 14C-labeled uridine. We found that the half-life of recently produced RNA increased from 4.0 days at 20°C to 15.8 days at 4°C, and 215 days at −4°C, while no degradation was detected at −18°C during a 1-year period. We discuss the implications of the strong temperature dependency of rRNA turnover for interpretation of microbiome data based on rRNA isolated from environmental samples.

Ribosomal RNA (rRNA) is used widely to investigate potentially active microorganisms in environmental samples, including soil microorganisms and other microbial communities that are subjected to pronounced seasonal variation in temperature. This raises a question about the turnover of intracellular microbial rRNA at environmentally relevant temperatures. We analyzed the turnover at four temperatures of RNA isolated from soil bacteria amended with 14C-labeled uridine. We found that the half-life of recently produced RNA increased from 4.0 days at 20°C to 15.8 days at 4°C, and 215 days at −4°C, while no degradation was detected at −18°C during a 1-year period. We discuss the implications of the strong temperature dependency of rRNA turnover for interpretation of microbiome data based on rRNA isolated from environmental samples. [ABSTRACT FROM AUTHOR]

Plastic particles are ubiquitous in marine systems and fragment into smaller pieces, such as nanoplastics (NPs). The effects of NPs on marine organisms are of growing concern but are not well understood. Marine sediments act as a sink for many contaminants, like microplastics, and are rich habitats for benthic micro- and meiofauna which are ecologically-important components of marine food webs; however, little is known about the sensitivities of specific organisms to NPs or the effects on community diversity and composition. Utilizing molecular methods, such as metabarcoding of environmental DNA/RNA, allows for the rapid and comprehensive detection of microscopic organisms via high-throughput sequencing to assess adverse effects at the community level. The objective of this study was to use a metabarcoding approach to investigate the effects of NPs on benthic micro- and meiofaunal community diversity. Mesocosms were created with sediment cores collected from the Narrow River estuary (Rhode Island, USA) and exposed to 900 nm diameter weathered polystyrene beads at concentrations of 0.1, 1, 10, or 100 mg/kg dry weight in sediment for two weeks. Following exposure, RNA and DNA were co-extracted from the sediment, RNA was reverse-transcribed, 18S and COI markers were PCR-amplified, and amplicons were sequenced on an Illumina MiSeq. Using the 18S marker and eRNA template, increases to α-diversity and significant differences to β-diversity were observed in the highest NP exposures relative to the control. Observed differences in community composition were driven by the differential abundance of several types of protists and arthropods. Significant dose-dependent shifts in composition were observed in β-diversity Jaccard and Unweighted-Unifrac metrics with the 18S marker using the RNA template. To our knowledge, this is the first demonstration of a dose-response relationship for NPs at a community level, and it highlights the value of using community-level endpoints to assess environmental impacts of nanoparticles. [Display omitted] • Nanoplastic alters benthic community composition and diversity. • Arthropods and unicellular eukaryotes are the most sensitive to nanoplastic. • Nanoplastic exposure resulted in dose-dependent effects at the community level. • eRNA metabarcoding is preferential to eDNA for community-level toxicology assessments. [ABSTRACT FROM AUTHOR]

Environmental DNA is increasingly being used in marine invasive species surveillance despite the inability to discriminate between contemporary intracellular (i.e., living) and extracellularly persistent (i.e., legacy) DNA fragments. Environmental RNA is emerging as a powerful alternative when distinguishing the living portion of a community is essential. A positive relationship between DNA and RNA signals may justify the use of DNA only for more rapid and cost-effective detections. In this study environmental DNA and RNA were co-extracted from settlement plates and water samples collected in an Auckland harbor, New Zealand. Samples were analyzed using a specific droplet digital PCR assay for the invasive Mediterranean fanworm (Sabella spallanzanii), combined with metabarcoding of metazoan communities (Cytochrome c oxidase subunit I). The number and magnitude of S. spallanzanii detections was higher in DNA compared to RNA, and in water samples. An assessment of detection sensitivity and specificity using a Receiver Operator Characteristics (ROC) analysis supported a relationship between the magnitude of DNA signal and the likelihood of RNA detection for both sampled matrices. A prediction threshold of 400 COI copies in DNA samples provides an indicator for the detection of eRNA, hence the putative presence of living S. spallanzanii population under the conditions tested in this study. Metabarcoding community analysis revealed the taxonomic composition of the water samples to be more diverse than the plate samples which were largely dominated by mollusks. There was a strong association between mollusks and presumed extracellular droplet digital PCR signals. Nevertheless, droplet digital PCR detection signals based on environmental DNA were negatively correlated with metabarcoding diversity indices on plates. This highlights complex interactions between environmental DNA and RNA detections and environmental matrices that can affect targeted approaches. These interactions need to be considered when designing surveillance programs.

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.

The ability to extract and purify messenger RNA directly from plants, decomposing organic matter and soil, followed by high-throughput sequencing of the pool of expressed genes, has spawned the emerging research area of metatranscriptomics. Each metatranscriptome provides a snapshot of the composition and relative abundance of actively transcribed genes, and thus provides an assessment of the interactions between soil microorganisms and plants, and collective microbial metabolic processes in many environments. We highlight current approaches for analysis of fungal transcriptome and metatranscriptome datasets across a gradient of community complexity, and note benefits and pitfalls associated with those approaches. We discuss knowledge gaps that limit our current ability to interpret metatranscriptome datasets and suggest future research directions that will require concerted efforts within the scientific community. [ABSTRACT FROM AUTHOR]

[Display omitted] • Sufficient amounts of fish eRNA for metabarcoding were present in water. • Sensitivity and positive predictivity of eRNA was better than those of eDNA. • eRNA can be used to identify false positives. • eRNA can act as a critical indicator of living biotic assemblages. Metabarcoding analysis using environmental DNA (eDNA) is increasingly being used for monitoring biota. However, knowledge regarding abundance of fish environmental RNA (eRNA) and its utility in ecological survey is limited. In this study, we aimed to perform eDNA/eRNA metabarcoding analyses of fishes using water samples from rivers in Japan. Sufficient amounts of eRNA from various species were present in the environmental water samples, which can support ecological survey based on eRNA metabarcoding. The sensitivity of eRNA was equivalent to that of eDNA; however, its positive predictivity was superior to that of eDNA. In addition, correlation with abundance in the ecological survey was higher for eRNA than for eDNA. Only eRNA (and the ratio of eRNA and eDNA) enabled distinguishing saltwater or brackish water fishes (false positives) that could be generated due to contamination from domestic wastewater containing their corpses or body parts or from bird feces. To the best of our knowledge, this is the first report of the possibility of eRNA to serve as a critical indicator of living biotic assemblages that actually inhabit sampling points. [ABSTRACT FROM AUTHOR]

• Current molecular methods have enhanced HABs/CHABs biomonitoring but have limitations. • CRISPR/Cas is a powerful and ultrasensitive molecular tool for molecular diagnosis. • Cas12/Cas13 effectors have potential as next-generation biomonitoring tools. • Multiple genes in bloom forming species could be targetted for CRISPR/Cas-based biomonitoring. Research on harmful algal and cyanobacterial blooms (HABs and CHABs) has risen dramatically due to their increasing global distribution, frequency, and intensity. These blooms jeopardize public health, ecosystem function, sustainability and can have negative economic impacts. Numerous monitoring programs have been established using light microscopy, liquid chromatography coupled to mass spectrometry (LC-MS), ELISA, and spectrophotometry to monitor HABs/CHABs outbreaks. Recently, DNA/RNA-based molecular methods have been integrated into these programs to replace or complement traditional methods through analyzing environmental DNA and RNA (eDNA/eRNA) with techniques such as quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), sandwich hybridization assay (SHA), isothermal amplification methods, and microarrays. These have enabled the detection of rare or cryptic species, enhanced sample throughput, and reduced costs and the need for visual taxonomic expertise. However, these methods have limitations, such as the need for high capital investment in equipment or detection uncertainties, including determining whether organisms are viable. In this review, we discuss the potential of newly developed molecular diagnosis technology based on Clustered Regularly Interspaced Short Palindromic Repeats/Cas proteins (CRISPR/Cas), which utilizes the prokaryotic adaptative immune systems of bacteria and archaea. Cas12 and Cas13-based platforms can detect both DNA and RNA with attomolar sensitivity within an hour. CRISPR/Cas diagnostic is a rapid, inexpensive, specific, and ultrasensitive technology that, with some further development, will provide many new platforms that can be used for HABs/CHABs biomonitoring and research. [Display omitted] [ABSTRACT FROM AUTHOR]

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive. • Ingested dsRNA spreads via hemolymph and is secreted in worker and royal jellies • Functional dsRNA flows horizontally among honey bees by jelly consumption • Worker and royal jellies harbor differential natural ssRNA and dsRNA populations • Diverse pathogenic RNA fragments naturally occur in worker and royal jellies RNA mobility among cells has been documented in plants and animals. Maori et al. show that RNA spreads further in honey bees and is horizontally transferred between individuals and across generations. Their findings demonstrate a transmissible RNA pathway with potential roles in social immunity and epigenetic communication between honey bees. [ABSTRACT FROM AUTHOR]

RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown. Here, we show that worker and royal jellies harbor robust RNA-binding activity. We report that a highly abundant jelly component, major royal jelly protein 3 (MRJP-3), acts as an extracellular non-sequence-specific RNA-aggregating factor. Multivalent RNA binding stimulates higher-order assembly of MRJP-3 into extracellular ribonucleoprotein granules that protect RNA from degradation and enhance RNA bioavailability. These findings reveal that honeybees have evolved a secreted dietary RNA-binding factor to concentrate, stabilize, and share RNA among individuals. Our work identifies high-order ribonucleoprotein assemblies with functions outside cells and organisms., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.