Available online at www.sciencedirect.com Improving the assessment of ecosystem and wildlife health: microbiome as an early indicator Maria Puig Ribas 1, Manuel García-Ulloa 2, Johan Espunyes 1 and ]] ]]]]]] ]] Oscar Cabezón 1 Human activities are causing dramatic declines in ecosystem estimated to be 100–1000 times higher than historical health, compromising the functioning of the life-support rates and it is estimated to further increase by system, economic activity, and animal and human health. In this 10–100 times over the next 50 years [3]. These human context, monitoring the health of ecosystems and wildlife impacts are causing dramatic declines in ecosystem populations is crucial for determining ecological dynamics and health, compromising the functioning of the life-support assessing management interventions. A growing body of system, the sustainment of economic activity, and an­ evidence indicates that microbiome provides a meaningful early imal and human health [4]. It is now clear that we cannot indicator of ecosystem and wildlife health. Microbiome is escape the responsibility and necessity of restoring and ubiquitous and both environmental and host-associated managing our planet. In this context, monitoring the microbiomes rapidly reflect anthropogenic disturbances. health of ecosystems and wildlife populations is crucial However, we still need to overcome current limitations such as for determining ecological dynamics, prioritizing active nucleic acid degradation, sequencing depth, and the management, and assessing the efficacy of management establishment of baseline data to maximize the potential of interventions. Nevertheless, the majority of health as­ microbiome studies. sessments and symptoms of ecosystem dysfunction are retrospective. Despite years of research, we still rely Addresses heavily on alterations that manifest late in the dis­ 1 Wildlife Conservation Medicine Research Group (WildCoM), turbance process such as changes in animal abundance Departament de Medicina i Cirurgia Animals, Universitat Autònoma de and richness [5], loss of interactions and functional di­ Barcelona, 08193 Bellaterra, Catalonia, Spain 2 Departament de Genètica i Microbiologia, Universitat Autònoma de versity [6,7], decline in ecosystem services [8], or pa­ Barcelona, 08193 Bellaterra, Catalonia, Spain thogen emergence [9,10]. We urgently need to develop reliable prospective indicators of wildlife and ecosystem Corresponding author: Cabezón, Oscar (oscar.cabezon@uab.cat) health with early-warning capabilities. One such pro­ mising indicator is the microbiome, described as the Current Opinion in Biotechnology 2023, 81:102923 interacting community of commensal, symbiotic, and This review comes from a themed issue on Environmental pathogenic microorganisms within a body space or other Biotechnology environment, together with the whole spectrum of mo­ lecules that they produce and mobile genetic elements Edited by Jaime Martinez Urtaza and Luis De León (i.e. phages, viruses, and extracellular DNA) [11]. For complete overview of the section, please refer to the article collection, “Environmental Biotechnology (2023)” Microbiome reflects ecosystem disturbance Available online 28 March 2023 A growing body of research points out that microbiome https://doi.org/10.1016/j.copbio.2023.102923 provides a meaningful early indicator of ecosystem dis­ 0958–1669/© 2023 The Author(s). Published by Elsevier Ltd. This is turbance [12]. First, microbiome is ubiquitous in ter­ an open access article under the CC BY license (http:// restrial, freshwater, and marine habitats, as well as in the creativecommons.org/licenses/by/4.0/). animals and plants living in them. Therefore, unlike most traditional indicators (e.g. macroinvertebrate com­ munities), microbiome approaches are potentially ap­ plicable to any ecosystem, including extreme or highly degraded environments. In ecosystems, environmental microbiome is a key component performing essential Introduction ecological processes such as primary production, nutrient Earth’s ecosystems are largely and increasingly domi­ cycling, and retention, and influencing plant diversity nated by humans through land transformation, resource and host-associated microbiomes [13–15]. exploitation, modifications in biodiversity, climate change, and alterations in biogeochemical cycles [1]. Crucial for signaling ecosystem health, microbiome is Particularly, the loss of biodiversity has been identified highly sensitive to environmental disturbances, including as one of the main threats and challenges for the coming anthropogenic impacts [16]. Recently, Rillig et al. [17] decades [2]. The current rate of species extinction is www.sciencedirect.com Current Opinion in Biotechnology 2023, 81:102923 2 Environmental Biotechnology showed that the complexity, composition, and overall Figure 1 abundance of soil microbiomes declined along with the number of global change factors, including resource availability, abiotic factors, toxic compounds, and micro­ plastics. Soil microbiomes have also shown to shift in re­ sponse to anticipated consequences of climate change, including elevated atmospheric carbon dioxide, elevated temperature, permafrost thawing, drought, floods, and wildfires [18]. One of the primary anthropogenic stressors driving ecosystem degradation worldwide is habitat loss as a consequence of land use change [19], which also influ­ ences the soil microbiome [20]. Detectable changes in environmental microbiomes of terrestrial, freshwater, and marine ecosystems are also induced by pollution through chemicals and light [21], mine drainage [22], oil spills [23], heavy metals [24], microplastics [25], and sewage waters Current Opinion in Biotechnology from urban centers [26]. The latter is not only an example of pollution but also illustrates how the encroachment of Graphical representation of microbiome dynamics. The horizontal human and domestic animal microbiomes and pathogens streaky bar represents normal oscillations or baseline data over time for can alter environmental microbiomes. Biological inva­ a particular microbiome metric (e.g. diversity). Disturbance events (orange vertical bar) can alter environmental conditions and trigger shifts sions, by microorganisms, plants, or animals, are one of in microbiome (solid black line), leading to alternative stable states that the main drivers of global change and generate, as well, significantly deviate from the natural state. Resistance is the ability of a shifts in the microbiome of native ecosystems [27]. community to withstand a disturbance without change. Resilience is the rate at which the original state is regained after a disturbance event. Only after the establishment of these baseline intervals of normality, we The examples above demonstrate that environmental can begin to understand microbiome responses to disturbance or microbiomes are generally not resistant to anthropogenic stress. disturbances. However, microbiomes could be resilient to environmental perturbations, quickly recovering their initial composition and hampering our ability to detect major outbreaks of emerging diseases in humans [33]. transient shifts (Figure 1). Encouragingly, Allison and The majority of emerging infectious disease threats Martiny [28] showed that, within a few years, microbial originate from wildlife, and outbreak risk is increased by composition still differed from that of undisturbed ecological degradation [9,34]. Over recent years, some communities, thus allowing realistic sampling intervals progress has been made in monitoring wildlife micro­ for ecosystem monitoring. Furthermore, microbial com­ biomes in order to predict, prevent, and control zoonotic munities are probably not functionally redundant and disease emergence. For example, in 2009, the U.S disturbances often impact the rates of ecosystem pro­ Agency for International Development launched the cesses [28]. This suggests that we can detect shifts in Emerging Pandemic Threats Program (EPT-1), which community composition and function as two com­ has been followed by a new generation of investments plementary axes of variation [29]. Overall, conditions (EPT-2, https://www.usaid.gov/ept2). Nevertheless, im­ altering the composition of environmental microbiomes portant steps still need to be taken to strengthen the may severely impact the structure, function, and natural monitoring of population trends and health for wild re­ balance of ecosystems, and increase human and animal servoir species, to identify novel infectious agents, and exposure to pathogens. Despite the evidence that mi­ to better understand the links between ecosystem de­ crobiome can be used to detect change processes in gradation and zoonotic spillover. ecosystems, its use as an indicator has mainly been evaluated for marine ecosystems, particularly for coral Concurrently, infectious diseases have been identified as reef ecosystems [30–32]. Therefore, microbiome still a growing threat to wildlife conservation, contributing to holds an unexplored diagnostic capacity to predict eco­ the global loss of biodiversity [35,36]. Amphibian chy­ system alterations and guide management interventions, tridiomycosis is perhaps the most striking example of the especially for terrestrial ecosystems. impact of diseases on biodiversity. Since its discovery in the 1990s, chytridiomycosis has been directly linked to the decline of over 500 amphibian species, the extinc­ Wildlife population health and bioindicator tion of 90, and continues to threaten amphibian diversity species globally [37]. Disease threats to biodiversity share Tracking the microbiome of wildlife populations has common anthropogenic drivers with disease outbreaks in potential human, animal, and ecosystem health appli­ humans [35]. Despite the need to monitor the health of cations. In an era of global change, we are witnessing declining wild populations, little progress has been made Current Opinion in Biotechnology 2023, 81:102923 www.sciencedirect.com Microbiome as indicator of ecosystem health Ribas et al. 3 on the use of microbiome as an indicator of wildlife biomonitor of the health of coastal marine ecosystems, population health and most pathogen threats still remain since corals are the backbone of reef systems, provide undiscovered. food and shelter for many species, and are tied to the changes in reef health [32]. The gut microbiome of Microbiome has been linked to metabolic functions, several filter-feeding species is especially sensitive to the aiding digestion, nutrition, development, and immune environment and also holds potential for aquatic eco­ defenses in animal hosts [15,38,39]. Shifts in microbial systems [45]. In terrestrial ecosystems, honeybee mi­ community composition may thus lead to dysfunction of crobiomes have been suggested as good proxies for host machinery, contributing to the development of a ecosystem health status [46]. Other promising indicators broad range of infectious and noninfectious diseases. are top predators or species with narrow ecological ni­ Animals interact with their environment extensively and ches as they can indicate changes in the trophic network environmental characteristics strongly shape microbiome and respond rapidly to minimal shifts in habitat condi­ composition and influence microbiome functionality. tions [41]. Bats are another interesting taxon that, de­ Anthropogenic disturbances also leave traceable changes spite having received great interest as reservoirs for in hosts’ microbiomes, suggesting that host-associated zoonotic agents, have not been explored in ecosystem microbiomes may be realistic indicators of individual and health assessments. Their diversity of species, broad population health in wildlife species. In amphibians, distribution, feeding habits, and ability to fly point out shifts in skin microbiome as a result of habitat de­ their inherent capacity to signal ecosystem alterations. In gradation and environmental pollutants can increase the fact, new studies demonstrate that the fecal bacterial susceptibility and the outcomes of disease [40]. Simi­ microbiome of insectivorous bats can reflect habitat de­ larly, impacts on the fecal microbiome of polar bears gradation and propose innovative uses of microbiome have been detected as a result of global change, sug­ monitoring [47]. gesting that it could be incorporated into health assess­ ments for conservation practices [41]. In honeybees, changes in the structure and function of the gut micro­ Future needs for microbiome studies biome were detected after pesticide exposure, which Since the advent of next-generation sequencing tech­ appeared to reflect the overall health of the colony [42]. nologies, microbiome studies rely on metagenomics to Moreover, monitoring microbiomes could significantly collect taxonomic and functional data from microbial improve ethical and practical aspects of wildlife sam­ communities [48]. These molecular technologies make pling, replacing procedures such as physical and che­ studying microbial communities a cost-effective alter­ mical restraint, blood sampling, or tissue samples (e.g. native to traditional methods and enable a more in­ toe clipping in amphibians). Indeed, microbiome studies tegrative approach. However, a careful balancing act can be performed in noninvasive samples, and in elusive, between resource allocation, sample size, and sequen­ protected, too-large, or too-small species [15]. Im­ cing depth must be performed in order to achieve portantly, these microbiome alterations seem to precede meaningful results. First, a large amount of genetic clinical manifestations of disease processes. For ex­ material should be recovered in order to achieve a ample, despite healthy appearance, coral microbiome complete community sampling. With variable results, communities under anthropogenic stress resembled multiple buffer solutions and rapid freezing, often in those of diseased individuals, potentially being at a tip­ liquid nitrogen, are the two most common methods ping point toward disease [43]. This shows that wildlife implemented in order to avoid high degrees of nucleic microbiomes could serve as a reliable early-warning acid degradation in fresh samples [49,50]. Even in the system, advising management interventions before dis­ best-case preservation scenario, partial fragmentation ease outbreaks or further biodiversity losses. and loss of genetic material are inevitable at the nucleic acid extraction step regardless of the method [51,52]; More broadly, wildlife microbiomes can also be used to commercial kits are fairly standardized and scalable but signal ecosystem degradation and inform overall eco­ provide moderate-to- low yields, while phase separation system health. Host-associated microbiomes are influ­ methods such as phenol–chloroform generate high yields enced by environmental conditions and can reflect but require manual precision and often carry con­ stressors and processes of change. It is clear, however, taminants to downstream processes. Second, sufficiently that not all wildlife species are equally capable of sig­ high sequencing depth must be met to provide a full naling ecosystem health. Therefore, we need to identify recovery of the community, a need that rapidly increases suitable bioindicator and biomonitor species, which re­ with the amount and diversity of the target sequences spond predictably to environmental change and quality [53]. The question of how much sequencing depth is [44]. Wildlife species that have disproportionate effects enough is often difficult to answer, particularly for ex­ on the ecosystem relative to their abundance are inter­ ploratory studies targeting low-diversity taxa and those esting targets since they may respond earlier to changes. involving nonmodel organisms, given the lack of re­ Coral microbiomes can be one such bioindicator/ ference studies. Thus, sequencing the totality of the www.sciencedirect.com Current Opinion in Biotechnology 2023, 81:102923 4 Environmental Biotechnology genetic material may only be suitable for select cases of study, since viruses do not have a universal marker gene high-quality, low-diversity samples with previous refer­ and are often found in low abundance in healthy in­ ences. Despite its known methodological biases [54], dividuals [57]. Targeted sequencing of functional genes researchers have turned to selectively sequence a gene is also tricky, as there are not many standardized kits or small subset of genes that serve as a proxy for the available and the scope of the intended scientific ques­ entire community composition or a subset of its func­ tions should be rather narrow by design. Recently, Na­ tionality. For instance, most taxonomic studies focus on nopore adaptive sequencing emerged as a promising tool deep amplicon sequencing of the 16S rRNA gene from for the study of low-abundance sequences through var­ prokaryotes, which nowadays can easily be recovered in ious enrichment approaches [58], although its full po­ its entirety through long-read sequencing technologies tential is still under study and development [59,60] [55,56]. Contrastingly, viral diversity is often difficult to (Figure 2). Figure 2 Current Opinion in Biotechnology Common drawbacks and constraints in the collection of nucleic acid data from environmental samples. The composition of microbial community samples becomes altered when subjected to laboratory procedures. For instance, nucleic acid material often gets fragmented when stored, and further degraded and partially lost during the extraction step. In exploratory studies, the decision of which type of sequencing to use is strongly constrained by the complexity of the sample and the resources available to afford the necessary sequencing depth to achieve meaningful results. Current Opinion in Biotechnology 2023, 81:102923 www.sciencedirect.com Microbiome as indicator of ecosystem health Ribas et al. 5 Besides methodological constraints, the use of micro­ equally valuable as an indicator of improvement in biome indicators still entails some other relevant lim­ health parameters. Particularly, microbiome could help itations. Microbiome responses to stress do not have a monitor the outcome of management interventions to straightforward interpretation and we cannot infer health restore ecosystem functionality or re-establish healthy or disease based solely on the direction of the change wild animal populations. Finally, further challenges re­ (i.e. increases or reductions in abundance, diversity, and main for the broadscale application of microbiome as a function). There is a growing consensus that occurrence health indicator, particularly in remote areas or devel­ and in some cases even degree of anthropogenic dis­ oping countries. Metagenomic tools require advanced turbance often manifests as one of the following micro­ technical and computational skills and, despite they biome alterations: 1) overall decrease in alpha-diversity, have become cheaper over the past years, they still entail 2) increase in beta-diversity, 3) decrease in beneficial significant costs. Therefore, these methods may not be and/or increase in pathogenic or disease-associated bac­ readily available in remote areas and can be prohibitively teria (sometimes can occur along an increase in alpha- expensive in low-income countries [72]. Moreover, in diversity), 4) increase in the dominance of generalist order to detect changes in microbiome indicators, it is over specialist bacteria, and 5) increase in the abundance likely that long-term datasets are needed, requiring long- of microbial resistance genes [16,40,61–69]. These term commitment and funding, and being even more parameters and changes may represent useful micro­ difficult to achieve in such regions. Unfortunately, most biome indicators of wildlife and ecosystem health. biodiversity hotspots and the highest rates of environ­ However, the magnitude and direction of these shifts are mental change occur in such remote or economically difficult to generalize across different ecosystems, re­ poorer regions, hampering many real-world applications sulting in both increases and reductions of particular for microbiome studies. species, microbial biomass, and activity [18]. A clear example is seen in arctic and subarctic ecosystems ex­ Conclusion periencing permafrost thawing where increases in alpha- Human activities are having dramatic impacts diversity are commonly reported [18,70]. Most probably, throughout the planet and we urgently need reliable this means that we need to develop specific microbiome early-warning indicators for monitoring the health of indicators and be aware of their particular responses in wildlife populations and ecosystems. Microbiome, both each ecosystem and species, and probably for each environmental and host-associated, can be one such threatening process. Local changes imply that local au­ promising indicator since it is ubiquitous, performs key thorities and environmental agencies should become processes in ecosystems and hosts, and reflects anthro­ more involved in microbiome studies, whereas now, they pogenic disturbances. Changes in the microbiome tend mostly attract the interest of international institutions. to occur earlier in the disturbance process compared with common health indicators, allowing effective monitoring Microbiomes naturally are dynamic, their composition of wildlife and ecosystem health as well as the outcomes and function oscillate within certain intervals, even in of management interventions. Nevertheless, micro­ undisturbed ecosystems and in healthy hosts [71]. biome potential as a health indicator remains largely Therefore, we need comprehensive analyses of the unexplored, mostly in terrestrial ecosystems. Important temporal and spatial variation of each unique microbial limitations still remain for the widespread application of community and the subsequent identification of base­ microbiome as a health indicator, including biotechno­ line reference data in order to correctly interpret mi­ logical tools, interpretation of shifts in selected para­ crobiome changes. Only after the establishment of these meters, understanding baseline and altered microbiome intervals of normality, we can begin to timely detect dynamics, and logistical constraints. Overcoming current microbiome responses to disturbance or stress (Figure 1). limitations will require establishing clear methods and Considering that human impacts are widespread best-guess baselines, increasing funding, and fostering throughout the planet, are we too late to establish international interdisciplinary collaboration. Future ef­ baseline data for the microbiomes of wildlife hosts and forts in this direction will maximize the potential of ecosystems? Probably. However, we can define health microbiome for monitoring wildlife and ecosystem based on reference data by comparing different ecosys­ health. tems and wildlife populations. Systems capable of maintaining ecological integrity, ecological processes, and services can act as proxies of pristine and un­ CRediT authorship contribution statement perturbed communities, and will constitute our best- Maria Puig Ribas: Conceptualization; Data curation; guess estimations for baseline microbiomes. Importantly, Writing – original draft; Writing – review & editing. the dynamic nature of microbiomes also allows for sig­ Manuel García-Ulloa: Writing – original draft. Johan naling positive changes in ecosystem and wildlife health. Espunyes: Methodology; Writing – original draft; Writing Most scientific attention has focused on studying mi­ – review & editing. Oscar Cabezón: Conceptualization; crobiome responses to disturbance, however, it may be Supervision; Validation; Writing – review & editing. www.sciencedirect.com Current Opinion in Biotechnology 2023, 81:102923 6 Environmental Biotechnology Data Availability ecosystem health status: potential and limitations. Appl Microbiol Biotechnol 2019, 103:6407-6421. No data were used for the research described in the ar­ 13. Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, Djahanschiri B, Zeller G, Mende DR, Alberti A, et al.: ticle. Structure and function of the global ocean microbiome. Science 2015, 348:1261359. Conflict of interest statement 14. Wagg C, Bender SF, Widmer F, Van Der Heijden MGA: Soil biodiversity and soil community composition determine ecosystem multifunctionality. Proc Natl Acad Sci 2014, The authors of the paper have no conflicts of interest to 111:5266-5270. disclose. All authors read, revised, and approved the final 15. Sehnal L, Brammer-Robbins E, Wormington AM, Blaha L, Bisesi J, paper. We confirm that the submitted paper has not • Larkin I, Martyniuk CJ, Simonin M, Adamovsky O: Microbiome composition and function in aquatic vertebrates: small been published or neither submitted for publication organisms making big impacts on aquatic animal health. Front elsewhere. Microbiol 2021, 12:567408. This paper synthesizes research on the microbiota of aquatic verte­ brates and discusses the impact of emerging stressors using an in­ tegrative approach by considering both host-associated and free-living Acknowledgements microbiota within an ecosystem. M.P.R. was funded through the 2021 FI Scholarship, Departament de Recerca i Universitats, Generalitat de Catalunya, Spain (FI_B 00171). 16. Rocca JD, Simonin M, Blaszczak JR, Ernakovich JG, Gibbons SM, •• Midani FS, Washburne AD: The Microbiome Stress Project: toward a global meta-analysis of environmental stressors and References and recommended reading their effects on microbial communities. Front Microbiol 2019, 10:3272. Papers of particular interest, published within the period of review, have This paper presents the Microbiome Stress Project, an open-access been highlighted as: database to facilitate cross-study analyses of microbiome responses to stressors. 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