- Email: [email protected]
Can Environmental RNA Revolutionize Biodiversity Science?
Trends in Ecology & Evolution
an individual to engage in biodiversity conservation behaviors are either difficult to identify or can take a long time to emerge [5]. Biodiversity issues are often diffuse, making the link between behavior and biodiversity impact difficult to examine. Furthermore, conservation communications often have varied goals; for example, raising issue awareness or soliciting donations. A few themes have emerged from the notable efforts towards guiding the strategic communication of conservation messages [6]. One of them is an enthusiasm for conservation optimism to combat the disengagement associated with negative messages as described by McAfee et al. [7] and to unite people to act. This is an example of message framing, which has been shown to be an effective tool for influencing behavior, attitudes, and judgment across disciplines from policy to psychology. Framing theory suggests that the structure or organization of a message can alter perception of its content [8]. However, the use of differently framed messages is just one of many factors known to influence how people respond to communications. Other factors include, but are not limited to, efficacy (both personal and collective), the messenger, social context, and the message channel; it is often a complex interaction between many of these factors [9,10]. These factors help us define the types of research questions that need to be answered to apply strategic messaging to conservation issues, but too often we do not subject conservation communications questions to the same rigor used to answer other applied questions. Developing effective message framing strategies will require multidisciplinary and methodologically varied approaches [11] and will take some conservationists into unfamiliar territory. Thankfully, suitable experimental frameworks exist and 694
are immediately practicable. Experimental frames should be made as analogous as possible and it is useful to test comprehension and the extent to which different frames convey the intended central idea [12]. In addition, studies should compare ways of talking about social issues that already exist in the public domain [12], so conservation optimism versus pessimism is a pragmatic starting point. McAfee and Connell point out that the line between a pessimistic and an optimistic message can be unclear; regardless, this should not deter efforts to build a corpus of experimental evidence with the aid of existing experimental frameworks. Using rigorous approaches, we can compile the empirical evidence that conservationists should start using as a matter of course when designing communications.
References 1. Kidd, L.R. et al. (2019) Neither hope nor fear: empirical evidence should drive biodiversity conservation strategies. Trends Ecol. Evol. 34, 278–282 2. McAfee, D and Connell, S.D. (2019) Balancing the benefits of optimism and pessimism in conservation: a response to Kidd, Bekessy and Garrard. Trends Ecol. Evol 34, 692–693 3. Kidd, L.R. et al. (2019) Messaging matters: a systematic review of the conservation messaging literature. Biol. Conserv. 326, 92–99 4. Veríssimo, D. et al. (2019) Conservation marketing as a tool to promote human-wildlife coexistence. In: Frank, B., et al. (Eds.), Human–Wildlife Interactions: Turning Conflict into Coexistence. Cambridge University Press, pp. 335–354 5. Se linske , M. et a l. ( 2 01 8 ) Re vi s i t i n g t he p ro m i s e of conservation psychology. Conserv. Biol. 32, 1 46 4 –14 6 8 6. Green, K.M. et al. (2019) A meta-analysis of social marketing campaigns to improve global conservation outcomes. Soc. Mar. Q. 25, 69–87 7. McAfee, D. et al. (2019) Everyone loves a success story: optimism inspires conservation engagement. Bioscience 69, 274–281 8. Entman, R.M. (1993) Framing: toward clarification of a fractured paradigm. J. Commun. 43, 51–58 9. Noar, S.M. (2006) A 10-year retrospective of research in health mass media campaigns: where do we go from here? J. Health Commun. 11, 21–42 10. White, K. et al. (2019) How to SHIFT consumer behaviors to be more sustainable: a literature review and guiding framework. J. Mark. 83, 22–49 11. Manuel, T. and Davey, L. (2009) Strategic frame analysis: providing the “evidence” for evidence-based communications. New Dir. Youth Dev. 2009, 29–38 12. Manuel, T. (2009) Who says your frames are better than mine? Making the case for strategic framing by using the power of experimental research. New Dir. Youth Dev. 2009, 71–82
Lack of research about how to design strategic messages to benefit biodiversity is limiting our ability to measure and optimize their effectiveness. As empirical evidence emerges, it will be fascinating to see whether heuristics developed in other disciplines will hold for conservation, but there is reason to believe that it should not be taken for granted. Re- Forum search into effective messaging for conservation behavior change can, and should, be informed by evidence-based, systematic approaches from these other disciplines. Our initial focus on optimism and pessimism was intended as a practiMelania E. Cristescu 1,* cal starting point and we welcome more nuanced and broad investigation into ef- The use of environmental RNA fective messaging strategies to benefit (eRNA) for species identification biodiversity.
Can Environmental RNA Revolutionize Biodiversity Science?
1 Interdisciplinary Conservation Science Research Group, School of Global, Urban, and Social Studies, RMIT University, Melbourne, VIC 3000, Australia 2 National Environmental Science Program Threatened Species Recovery Hub, Australia
*Correspondence: [email protected] (L.R. Kidd). https://doi.org/10.1016/j.tree.2019.05.010 © 2019 Elsevier Ltd. All rights reserved.
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
remains unexplored due to the observation that in vitro RNA is much less stable than DNA. However, recent lines of evidence suggest that RNA may be abundantly excreted by organisms and sufficiently persistent in the environment to reconstruct community composition and gene expression.
Trends in Ecology & Evolution
Can eRNA Pick Up Where eDNA Fails? The prospect of using the DNA released by organisms into their environment as a sensitive and efficient method for species detection has been embraced with great enthusiasm [1,2]. By contrast, the possible use of environmental RNA (eRNA, see Glossary) markers for monitoring biodiversity trends has received little attention (but see [2–4]), mainly due to the observation that in vitro RNA is a much less stable molecule than DNA. Anyone who has performed an RNA extraction can attest to the lability of RNA. Thus, RNA persistence outside the organism is expected to be at most ephemeral. This assumption, however, remains largely untested, and does not consider the complex ‘ecology’ of RNA. I challenge this view here by discussing circumstantial evidence of the production, state, and detectability of eRNA in terrestrial and aquatic settings.
One of the major problems with current species-detection protocols based on environmental DNA (eDNA) is the high rate of false positives (the false detection of species not present in the sampled environment) and negatives (species undetected when present) [4]. False detections may occur due to contamination or inadequate protocols, but can also be due to the natural transport of eDNA from one environment to another or because of the movement of eDNA from deeper sediments to superficial layers of sediments or the water column. Such natural processes can obscure spatial patterns of biodiversity or can impede the discrimination between past and current community assemblages. False negatives can also be due to natural processes of dilution and transport of eDNA from the original site to remote locations. The longer the molecule persists in the environment, the less discrete the spatiotemporal signal is.
A molecule with a higher production rate but a faster turnover could resolve some of these problems that are inherent in metabarcoding studies (Figure 1). Thus, eRNA-based markers could be better indicators of living biotic assemblages that are closely associated with the sampling site. This would be an important advance because distinguishing living organisms (metabolically active or dormant) from those that are dead is often very important in ecological surveys, particularly when estimating biosecurity risks [2,4]. Moreover, unlike eDNA, eRNA contains information on the gene expression profile of natural communities. Can eRNA meet these high standards?
Evidence from Paleogenetics and Forensics Although the term ancient RNA (aRNA) has only been sporadically used, several studies indicate that aRNA might be
Trends in Ecology & Evolution
Figure 1. The Ecology of Environmental RNA (eRNA). It is likely that RNA abundance and persistence in the environment depends on several factors: the rate and mechanism of release, molecular structure (i.e., length, conformation), mechanisms of transport (free, particle-bound, or association with cells, organelles, vesicular membranes, or capsid proteins), the abiotic environment (i.e., light, oxygen, pH, salinity, humidity, substrates), and the biotic environment (i.e., the species composition of the biotic assemblages, the activity of microbial community involved in decomposition, and the production of extracellular enzymes).
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
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Trends in Ecology & Evolution
more ubiquitous than was previously thought. aRNA has been successfully isolated from fossils (RNA viruses, yeast, plants, and mammals). The first attempts to sequence aRNA were inspired by the successful germination of ~2000 year old seeds, a process known to require intact RNA. Not only were scientists successful in sequencing aRNA from old seeds such as the 700 year old kernels of maize, but they also recovered surprisingly long fragments – longer than those of ancient DNA (aDNA) [5]. At about the time the first ancient viral genomes were successfully assembled [6], forensic laboratories started to use RNA as a means of identifying diseases, tissue type, and degree of degradation in post-mortem specimens. Gene expression profiling based on mRNA has also been used to make inferences about the mechanisms leading to death and/or time since death [7]. Thus, paleogenetic and forensic studies suggest that, at least under the right conditions, RNA can persist for long periods of time.
packed in retrovirus-like proteins which form a protective capsid, allowing efficient transport of mRNA from neurons to muscles. Although the full significance of these mRNA protection and transport mechanisms is not understood, there is mounting evidence that metabolically active organisms can release large amounts of RNA into the environment. Thus, it is intuitive to postulate that protective structures (cells, vesicles, capsids) are not only vehicles of transport but can also greatly extend the lifetime of RNA in the environment after its release from micro- or macro-organisms.
Evidence from Functional Genomics
Perhaps even more unexpected is the evidence that eRNA can have complex functions outside the progenitor organism. Gene silencing can be induced by exposure to double-stranded RNA (dsRNA) and miRNA encountered in the environment, both of which can be surprisingly stable. Thus mechanism, known as environmental RNA interference (eRNAi), has been Evidence from Cellular Biology Recent developments in cellular biology documented in many plants and inverteprovide an in-depth perspective on the brates [11]. Thus environmentally triggered state and persistence of RNA excreted gene regulation appears to be common, by cells. Almost all prokaryotic and eu- suggesting that eRNA has a role in the comkaryotic cells are capable of producing munication between organisms: identifying extracellular vesicles (EVs). Such vesi- pathogens, crossregulation of gene exprescles have long been considered to be sion in organisms with symbiotic relationwaste disposed of by the cells. Only ships, etc. Such studies provide further recently has it been recognized that evidence in support of the presence of cells can communicate with neighboring functionally potent RNA molecules in the or distant cells by using molecules environment. packed and transported in EVs [8]. These vesicles act as natural carriers of Evidence from Metagenomics and RNA, including mRNA and microRNA Metabarcoding (miRNA), that provide a stable environ- The term eRNA is most frequently enment preventing RNA digestion by RN- countered in metagenomics and metaases [9]. Unexpectedly high abundances barcoding studies. However, most studies and diversity of EVs are present in use bulk samples of unsorted organisms, body fluids that often leak into the extracting most of the RNA directly from environment. Cells have several other live organisms. The inadvertent use of vehicles for delivering stable RNA to dis- the term eRNA in such studies generates tant locations. A recent study by [10] confusion because the RNA extracted is revealed that Drosophila mRNA can be expected to include mostly organismal 696
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
Glossary Ancient DNA (aDNA): DNA isolated from old specimens or from various environments in the absence of obvious fossilized remains of organisms. Such DNA is usually considered to be of low quality, and requires special techniques and sterile conditions for extraction and amplification. Ancient RNA (aRNA): RNA isolated from fossilized organisms (viruses, seeds, mammals). It is much less well studied and understood than aDNA. Double-stranded RNA (dsRNA): RNA with two complementary strands, similar to DNA but with the replacement of thymine by uracil. dsRNA can trigger gene silencing in eukaryotes by a process known as RNA interference (RNAi). Environmental DNA (eDNA): refers to traces of DNA released by organisms into their environment. eDNA can be collected not only from environmental samples (water, soil, or air) as extracellular DNA but also as cellular DNA (fragments of tissue, gametes) recently released into the environment. Environmental RNA (eRNA): refers to RNAs released by organisms into their surrounding environment. The term is used here to refer strictly to RNA extracted from the environment (in cellular, vesicular, or free form) in the absence of progenitor organisms. The term eRNA is used inadvertently in the metabarcoding literature to refer to RNA extracted from bulk, unsorted biological material (e.g., bulk zooplankton samples or bulk benthic samples of micro- or macroinvertebrates). Environmental RNA interference (eRNAi): refers to sequence-specific gene silencing that crosses cellular boundaries when organisms encounter environmental dsRNA. Environmental transcriptomics: also known as metatranscriptomics, the term often refers to sequencing mRNA from a bulk sample of microbes generating the community gene expression. The term is used here to refer strictly to gene expression profiling based on eRNA collected in the absence of organisms, a non-invasive approach that could apply to both eukaryotes and prokaryotes, and span all trophic levels. Expression profiling: measuring the activity of all genes present in a particular sample (cell, tissue, or whole organism), providing information on their function. Extracellular vesicles (EVs): cell-derived membranous structures containing lipids, proteins, sugars, and nucleic acids, particularly RNAs. Cells from all domains of life produce EVs that are involved in multiple processes of cell-to-cell communication as well as interspecific signaling. EVs may also offer protection to RNA against extracellular enzymes. Metabarcoding: a DNA-based identification method involving the extraction of multiple species from a mass collection of specimens (bulk samples) or from complex and possibly degraded samples of eDNA. The metabarcoding approach is also known as amplicon sequencing and is applied frequently to microbial communities, but can be also applied efficiently to meio- and megafauna.
Trends in Ecology & Evolution
Metagenomics: the study of microbial communities sampled directly from their environment by examining the genomic compositions of all organisms rather than examining the genomes of separate species. MicroRNA (miRNA): small noncoding RNA with a role in gene silencing. Such molecules can be surprisingly stable owing to their ability to bind to proteins and subcellular compartments.
Box 1. Important Questions for Future Research on eRNA eRNA Origin The release of RNA into the environment (water or soil) is poorly understood. As with eDNA, the shedding of cells (via body fluids and remains) could be an important source of eRNA leakage into the environment. However, other means of active RNA excretion could be equally relevant. Q1. What are the major sources of eRNA? eRNA State With respect to its molecular state, RNA is a much more complex molecule that DNA. Presumably eRNA transitions from an intracellular or vesicular form to an extracellular or extravesicular form.
entirely new research field of environmental transcriptomics.
Q2. What is the state of eRNA in various environments? Q3. What are the conformational changes of RNA molecules released into the environment? Q4. How is extracellular RNA cycled in the environment?
Acknowledgments
eRNA persistence
I would like to thank J. Littlefair, X. Pochon, M. Harris, K. Kagzi, and K. Millette for stimulating discussions.
Although factors determining eDNA persistence are being explored, very little is known about the factors that might determine eRNA persistence.
This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair program.
Q5. What factors influence eRNA persistence? eRNA Degradation
1
Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada
Q6. What are the main exogenous and endogenous factors that influence RNA instability? Q7. What are the rates of RNA decay? Q8. How do RNA decay rates vary in different environments? Q9. What is the concentration of RNA in various environments?
*Correspondence: [email protected] (M.E. Cristescu). https://doi.org/10.1016/j.tree.2019.05.003
eRNA Applications
© 2019 Elsevier Ltd. All rights reserved.
Q10. Can eRNA be used in metabarcoding and metatranscriptomic studies?
both terrestrial and aquatic environments. The reason for its persistence outside the organismal environment is not understood (Figure 1), but several possible mechanisms could be invoked (e.g., protection by EVs, other remains of cellular compartments, protein capsids, or other binding molecules). Clearly, understanding the mechanisms of RNA release, transport, molecular state, and degradation rates is crucial for developing reliable metabarcoding markers (Box 1). The circumstantial evidence that eRNA might be less prone to false positives and negatives requires definitive confirmation. Perhaps more exciting is the prospect of using eRNA to develop functional markers for estimating gene expression activity for both prokaryotic and eukaryotic organisms, Concluding Remarks Although RNA is viewed as a very fragile and for investigating functional links across molecule that readily degrades in vitro, trophic levels. Such an approach would there is growing evidence that eRNA can allow assessment of the health of complex be abundant and surprisingly persistent in biotic assemblages by opening up an RNA and only traces of eRNA. Bulk extractions are commonly done in studies on microbial diversity because sampling the environment without sampling the organisms is often impossible. Nevertheless, metabarcoding studies based on bulk samples of DNA and RNA conclude that RNA often shows stronger relations with environmental variables than does DNA [12]. One recent study employing metabarcoding approaches on true environmental samples demonstrates that eRNA provides a slightly better correlation with morphological indices of diversity [2]. As a result, some authors recommend the complementary use of eDNA and eRNA in metabarcoding surveys [4,12].
References 1. Ficetola, G.F. et al. (2008) Species detection using environmental DNA from water samples. Biol. Lett. 4, 423–425 2. Pochon, X. et al. (2017) Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications. PLoS One 12, e0187636 3. Pawlowski, J. et al. (2018) The future of biotic indices in the ecogenomic era: Integrating eDNA metabarcoding in biological assessment of aquatic ecosystems. Sci. Total Environ. 637/638, 1295–1310 4. Cristescu, M.E. and Hebert, P.D.N. (2018) Uses and misuses of environmental DNA in biodiversity science and conservation. Annu. Rev. Ecol. Evol. Syst. 49, 209–230 5. Fordyce, S.L. et al. (2013) Deep sequencing of RNA from ancient maize kernels. PLoS One 8, e50961 6. Tumpey, T.M. et al. (2005) Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310, 77–80 7. Yasojima, K. et al. (2001) High stability of mRNAs postmortem and protocols for their assessment by RT-PCR. Brain Res. 8, 212–218 8. Sukhvinder, G. et al. (2019) Extracellular membrane vesicles in the three domains of life and beyond. FEMS Microbiol. Rev. 43, 273–303 9. Koga, Y. et al. (2011) Exosome can prevent RNase from degrading microRNA in feces. J. Gastrointest. Oncol. 2, 215–222 10. Ashley, J. et al. (2018) Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell 172, 262–274 11. Whangbo, J.S. and Hunter, C.P. (2008) Environmental RNA interference. Trends Genet. 24, 297–305Whangbo, J.S. and Hunter, C.P. (2008) Environmental RNA interference. Trends in Genetics 24, 297–305 12. Laroche, O. et al. (2016) First evaluation of foraminiferal metabarcoding for monitoring environmental impact from an offshore oil drilling site. Mar. Environ. Res. 120, 225–235
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an individual to engage in biodiversity conservation behaviors are either difficult to identify or can take a long time to emerge [5]. Biodiversity issues are often diffuse, making the link between behavior and biodiversity impact difficult to examine. Furthermore, conservation communications often have varied goals; for example, raising issue awareness or soliciting donations. A few themes have emerged from the notable efforts towards guiding the strategic communication of conservation messages [6]. One of them is an enthusiasm for conservation optimism to combat the disengagement associated with negative messages as described by McAfee et al. [7] and to unite people to act. This is an example of message framing, which has been shown to be an effective tool for influencing behavior, attitudes, and judgment across disciplines from policy to psychology. Framing theory suggests that the structure or organization of a message can alter perception of its content [8]. However, the use of differently framed messages is just one of many factors known to influence how people respond to communications. Other factors include, but are not limited to, efficacy (both personal and collective), the messenger, social context, and the message channel; it is often a complex interaction between many of these factors [9,10]. These factors help us define the types of research questions that need to be answered to apply strategic messaging to conservation issues, but too often we do not subject conservation communications questions to the same rigor used to answer other applied questions. Developing effective message framing strategies will require multidisciplinary and methodologically varied approaches [11] and will take some conservationists into unfamiliar territory. Thankfully, suitable experimental frameworks exist and 694
are immediately practicable. Experimental frames should be made as analogous as possible and it is useful to test comprehension and the extent to which different frames convey the intended central idea [12]. In addition, studies should compare ways of talking about social issues that already exist in the public domain [12], so conservation optimism versus pessimism is a pragmatic starting point. McAfee and Connell point out that the line between a pessimistic and an optimistic message can be unclear; regardless, this should not deter efforts to build a corpus of experimental evidence with the aid of existing experimental frameworks. Using rigorous approaches, we can compile the empirical evidence that conservationists should start using as a matter of course when designing communications.
References 1. Kidd, L.R. et al. (2019) Neither hope nor fear: empirical evidence should drive biodiversity conservation strategies. Trends Ecol. Evol. 34, 278–282 2. McAfee, D and Connell, S.D. (2019) Balancing the benefits of optimism and pessimism in conservation: a response to Kidd, Bekessy and Garrard. Trends Ecol. Evol 34, 692–693 3. Kidd, L.R. et al. (2019) Messaging matters: a systematic review of the conservation messaging literature. Biol. Conserv. 326, 92–99 4. Veríssimo, D. et al. (2019) Conservation marketing as a tool to promote human-wildlife coexistence. In: Frank, B., et al. (Eds.), Human–Wildlife Interactions: Turning Conflict into Coexistence. Cambridge University Press, pp. 335–354 5. Se linske , M. et a l. ( 2 01 8 ) Re vi s i t i n g t he p ro m i s e of conservation psychology. Conserv. Biol. 32, 1 46 4 –14 6 8 6. Green, K.M. et al. (2019) A meta-analysis of social marketing campaigns to improve global conservation outcomes. Soc. Mar. Q. 25, 69–87 7. McAfee, D. et al. (2019) Everyone loves a success story: optimism inspires conservation engagement. Bioscience 69, 274–281 8. Entman, R.M. (1993) Framing: toward clarification of a fractured paradigm. J. Commun. 43, 51–58 9. Noar, S.M. (2006) A 10-year retrospective of research in health mass media campaigns: where do we go from here? J. Health Commun. 11, 21–42 10. White, K. et al. (2019) How to SHIFT consumer behaviors to be more sustainable: a literature review and guiding framework. J. Mark. 83, 22–49 11. Manuel, T. and Davey, L. (2009) Strategic frame analysis: providing the “evidence” for evidence-based communications. New Dir. Youth Dev. 2009, 29–38 12. Manuel, T. (2009) Who says your frames are better than mine? Making the case for strategic framing by using the power of experimental research. New Dir. Youth Dev. 2009, 71–82
Lack of research about how to design strategic messages to benefit biodiversity is limiting our ability to measure and optimize their effectiveness. As empirical evidence emerges, it will be fascinating to see whether heuristics developed in other disciplines will hold for conservation, but there is reason to believe that it should not be taken for granted. Re- Forum search into effective messaging for conservation behavior change can, and should, be informed by evidence-based, systematic approaches from these other disciplines. Our initial focus on optimism and pessimism was intended as a practiMelania E. Cristescu 1,* cal starting point and we welcome more nuanced and broad investigation into ef- The use of environmental RNA fective messaging strategies to benefit (eRNA) for species identification biodiversity.
Can Environmental RNA Revolutionize Biodiversity Science?
1 Interdisciplinary Conservation Science Research Group, School of Global, Urban, and Social Studies, RMIT University, Melbourne, VIC 3000, Australia 2 National Environmental Science Program Threatened Species Recovery Hub, Australia
*Correspondence: [email protected] (L.R. Kidd). https://doi.org/10.1016/j.tree.2019.05.010 © 2019 Elsevier Ltd. All rights reserved.
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
remains unexplored due to the observation that in vitro RNA is much less stable than DNA. However, recent lines of evidence suggest that RNA may be abundantly excreted by organisms and sufficiently persistent in the environment to reconstruct community composition and gene expression.
Trends in Ecology & Evolution
Can eRNA Pick Up Where eDNA Fails? The prospect of using the DNA released by organisms into their environment as a sensitive and efficient method for species detection has been embraced with great enthusiasm [1,2]. By contrast, the possible use of environmental RNA (eRNA, see Glossary) markers for monitoring biodiversity trends has received little attention (but see [2–4]), mainly due to the observation that in vitro RNA is a much less stable molecule than DNA. Anyone who has performed an RNA extraction can attest to the lability of RNA. Thus, RNA persistence outside the organism is expected to be at most ephemeral. This assumption, however, remains largely untested, and does not consider the complex ‘ecology’ of RNA. I challenge this view here by discussing circumstantial evidence of the production, state, and detectability of eRNA in terrestrial and aquatic settings.
One of the major problems with current species-detection protocols based on environmental DNA (eDNA) is the high rate of false positives (the false detection of species not present in the sampled environment) and negatives (species undetected when present) [4]. False detections may occur due to contamination or inadequate protocols, but can also be due to the natural transport of eDNA from one environment to another or because of the movement of eDNA from deeper sediments to superficial layers of sediments or the water column. Such natural processes can obscure spatial patterns of biodiversity or can impede the discrimination between past and current community assemblages. False negatives can also be due to natural processes of dilution and transport of eDNA from the original site to remote locations. The longer the molecule persists in the environment, the less discrete the spatiotemporal signal is.
A molecule with a higher production rate but a faster turnover could resolve some of these problems that are inherent in metabarcoding studies (Figure 1). Thus, eRNA-based markers could be better indicators of living biotic assemblages that are closely associated with the sampling site. This would be an important advance because distinguishing living organisms (metabolically active or dormant) from those that are dead is often very important in ecological surveys, particularly when estimating biosecurity risks [2,4]. Moreover, unlike eDNA, eRNA contains information on the gene expression profile of natural communities. Can eRNA meet these high standards?
Evidence from Paleogenetics and Forensics Although the term ancient RNA (aRNA) has only been sporadically used, several studies indicate that aRNA might be
Trends in Ecology & Evolution
Figure 1. The Ecology of Environmental RNA (eRNA). It is likely that RNA abundance and persistence in the environment depends on several factors: the rate and mechanism of release, molecular structure (i.e., length, conformation), mechanisms of transport (free, particle-bound, or association with cells, organelles, vesicular membranes, or capsid proteins), the abiotic environment (i.e., light, oxygen, pH, salinity, humidity, substrates), and the biotic environment (i.e., the species composition of the biotic assemblages, the activity of microbial community involved in decomposition, and the production of extracellular enzymes).
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
695
Trends in Ecology & Evolution
more ubiquitous than was previously thought. aRNA has been successfully isolated from fossils (RNA viruses, yeast, plants, and mammals). The first attempts to sequence aRNA were inspired by the successful germination of ~2000 year old seeds, a process known to require intact RNA. Not only were scientists successful in sequencing aRNA from old seeds such as the 700 year old kernels of maize, but they also recovered surprisingly long fragments – longer than those of ancient DNA (aDNA) [5]. At about the time the first ancient viral genomes were successfully assembled [6], forensic laboratories started to use RNA as a means of identifying diseases, tissue type, and degree of degradation in post-mortem specimens. Gene expression profiling based on mRNA has also been used to make inferences about the mechanisms leading to death and/or time since death [7]. Thus, paleogenetic and forensic studies suggest that, at least under the right conditions, RNA can persist for long periods of time.
packed in retrovirus-like proteins which form a protective capsid, allowing efficient transport of mRNA from neurons to muscles. Although the full significance of these mRNA protection and transport mechanisms is not understood, there is mounting evidence that metabolically active organisms can release large amounts of RNA into the environment. Thus, it is intuitive to postulate that protective structures (cells, vesicles, capsids) are not only vehicles of transport but can also greatly extend the lifetime of RNA in the environment after its release from micro- or macro-organisms.
Evidence from Functional Genomics
Perhaps even more unexpected is the evidence that eRNA can have complex functions outside the progenitor organism. Gene silencing can be induced by exposure to double-stranded RNA (dsRNA) and miRNA encountered in the environment, both of which can be surprisingly stable. Thus mechanism, known as environmental RNA interference (eRNAi), has been Evidence from Cellular Biology Recent developments in cellular biology documented in many plants and inverteprovide an in-depth perspective on the brates [11]. Thus environmentally triggered state and persistence of RNA excreted gene regulation appears to be common, by cells. Almost all prokaryotic and eu- suggesting that eRNA has a role in the comkaryotic cells are capable of producing munication between organisms: identifying extracellular vesicles (EVs). Such vesi- pathogens, crossregulation of gene exprescles have long been considered to be sion in organisms with symbiotic relationwaste disposed of by the cells. Only ships, etc. Such studies provide further recently has it been recognized that evidence in support of the presence of cells can communicate with neighboring functionally potent RNA molecules in the or distant cells by using molecules environment. packed and transported in EVs [8]. These vesicles act as natural carriers of Evidence from Metagenomics and RNA, including mRNA and microRNA Metabarcoding (miRNA), that provide a stable environ- The term eRNA is most frequently enment preventing RNA digestion by RN- countered in metagenomics and metaases [9]. Unexpectedly high abundances barcoding studies. However, most studies and diversity of EVs are present in use bulk samples of unsorted organisms, body fluids that often leak into the extracting most of the RNA directly from environment. Cells have several other live organisms. The inadvertent use of vehicles for delivering stable RNA to dis- the term eRNA in such studies generates tant locations. A recent study by [10] confusion because the RNA extracted is revealed that Drosophila mRNA can be expected to include mostly organismal 696
Trends in Ecology & Evolution, August 2019, Vol. 34, No. 8
Glossary Ancient DNA (aDNA): DNA isolated from old specimens or from various environments in the absence of obvious fossilized remains of organisms. Such DNA is usually considered to be of low quality, and requires special techniques and sterile conditions for extraction and amplification. Ancient RNA (aRNA): RNA isolated from fossilized organisms (viruses, seeds, mammals). It is much less well studied and understood than aDNA. Double-stranded RNA (dsRNA): RNA with two complementary strands, similar to DNA but with the replacement of thymine by uracil. dsRNA can trigger gene silencing in eukaryotes by a process known as RNA interference (RNAi). Environmental DNA (eDNA): refers to traces of DNA released by organisms into their environment. eDNA can be collected not only from environmental samples (water, soil, or air) as extracellular DNA but also as cellular DNA (fragments of tissue, gametes) recently released into the environment. Environmental RNA (eRNA): refers to RNAs released by organisms into their surrounding environment. The term is used here to refer strictly to RNA extracted from the environment (in cellular, vesicular, or free form) in the absence of progenitor organisms. The term eRNA is used inadvertently in the metabarcoding literature to refer to RNA extracted from bulk, unsorted biological material (e.g., bulk zooplankton samples or bulk benthic samples of micro- or macroinvertebrates). Environmental RNA interference (eRNAi): refers to sequence-specific gene silencing that crosses cellular boundaries when organisms encounter environmental dsRNA. Environmental transcriptomics: also known as metatranscriptomics, the term often refers to sequencing mRNA from a bulk sample of microbes generating the community gene expression. The term is used here to refer strictly to gene expression profiling based on eRNA collected in the absence of organisms, a non-invasive approach that could apply to both eukaryotes and prokaryotes, and span all trophic levels. Expression profiling: measuring the activity of all genes present in a particular sample (cell, tissue, or whole organism), providing information on their function. Extracellular vesicles (EVs): cell-derived membranous structures containing lipids, proteins, sugars, and nucleic acids, particularly RNAs. Cells from all domains of life produce EVs that are involved in multiple processes of cell-to-cell communication as well as interspecific signaling. EVs may also offer protection to RNA against extracellular enzymes. Metabarcoding: a DNA-based identification method involving the extraction of multiple species from a mass collection of specimens (bulk samples) or from complex and possibly degraded samples of eDNA. The metabarcoding approach is also known as amplicon sequencing and is applied frequently to microbial communities, but can be also applied efficiently to meio- and megafauna.
Trends in Ecology & Evolution
Metagenomics: the study of microbial communities sampled directly from their environment by examining the genomic compositions of all organisms rather than examining the genomes of separate species. MicroRNA (miRNA): small noncoding RNA with a role in gene silencing. Such molecules can be surprisingly stable owing to their ability to bind to proteins and subcellular compartments.
Box 1. Important Questions for Future Research on eRNA eRNA Origin The release of RNA into the environment (water or soil) is poorly understood. As with eDNA, the shedding of cells (via body fluids and remains) could be an important source of eRNA leakage into the environment. However, other means of active RNA excretion could be equally relevant. Q1. What are the major sources of eRNA? eRNA State With respect to its molecular state, RNA is a much more complex molecule that DNA. Presumably eRNA transitions from an intracellular or vesicular form to an extracellular or extravesicular form.
entirely new research field of environmental transcriptomics.
Q2. What is the state of eRNA in various environments? Q3. What are the conformational changes of RNA molecules released into the environment? Q4. How is extracellular RNA cycled in the environment?
Acknowledgments
eRNA persistence
I would like to thank J. Littlefair, X. Pochon, M. Harris, K. Kagzi, and K. Millette for stimulating discussions.
Although factors determining eDNA persistence are being explored, very little is known about the factors that might determine eRNA persistence.
This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair program.
Q5. What factors influence eRNA persistence? eRNA Degradation
1
Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada
Q6. What are the main exogenous and endogenous factors that influence RNA instability? Q7. What are the rates of RNA decay? Q8. How do RNA decay rates vary in different environments? Q9. What is the concentration of RNA in various environments?
*Correspondence: [email protected] (M.E. Cristescu). https://doi.org/10.1016/j.tree.2019.05.003
eRNA Applications
© 2019 Elsevier Ltd. All rights reserved.
Q10. Can eRNA be used in metabarcoding and metatranscriptomic studies?
both terrestrial and aquatic environments. The reason for its persistence outside the organismal environment is not understood (Figure 1), but several possible mechanisms could be invoked (e.g., protection by EVs, other remains of cellular compartments, protein capsids, or other binding molecules). Clearly, understanding the mechanisms of RNA release, transport, molecular state, and degradation rates is crucial for developing reliable metabarcoding markers (Box 1). The circumstantial evidence that eRNA might be less prone to false positives and negatives requires definitive confirmation. Perhaps more exciting is the prospect of using eRNA to develop functional markers for estimating gene expression activity for both prokaryotic and eukaryotic organisms, Concluding Remarks Although RNA is viewed as a very fragile and for investigating functional links across molecule that readily degrades in vitro, trophic levels. Such an approach would there is growing evidence that eRNA can allow assessment of the health of complex be abundant and surprisingly persistent in biotic assemblages by opening up an RNA and only traces of eRNA. Bulk extractions are commonly done in studies on microbial diversity because sampling the environment without sampling the organisms is often impossible. Nevertheless, metabarcoding studies based on bulk samples of DNA and RNA conclude that RNA often shows stronger relations with environmental variables than does DNA [12]. One recent study employing metabarcoding approaches on true environmental samples demonstrates that eRNA provides a slightly better correlation with morphological indices of diversity [2]. As a result, some authors recommend the complementary use of eDNA and eRNA in metabarcoding surveys [4,12].
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