The epidermal ecotone: a proposed model system for marine viral ecology at the animal-environmental interface

Abstract

Marine animal epidermal surfaces act as a transitional viral habitat—an epidermal ecotone—between tissues and the environment, supporting diverse tripartite relationships between animals, microbes, and viruses, which have yet to be investigated. In this review, we synthesize the viral ecology of marine animal epidermal tissues, surfaces, and aura biomes to identify knowledge gaps and highlight the value of marine animal epidermis as a novel model system for microbiome research.

Introduction

Marine animals are unique microbial habitats that provide diverse substrates for colonization. The recruitment of commensal microbes as a competitive buffer against environmental pathogens is a successful immune strategy^1–4. A marine animal’s epidermis is in constant direct contact with the water column and therefore has a high potential to exchange microbes, viruses, and nutrients with the environment. The epidermal microbiome is a mix of taxa that have coevolved with the animal host and adapted to the corporeal niche plus additional taxa that are acquired from the environment throughout the animal’s life^5,6. Bacteria and archaea perform most of the biological processes in the microbiome, but viruses are significant drivers of microbiome functions^7,8.

In this review, we have grouped the marine animal virome into two categories of virus: animal viruses, which infect the animal host, and the animal’s microvirome, comprised of the viruses that infect the animal microbiome, including the viruses of micro-eukaryotes and archaea, and bacteriophages (phages) infecting bacteria. An animal virus in the microbiome has a simple niche: it either infects the animal and persists (virulently or latently) until the animal dies or the immune system removes it, or the virus does not infect the host and remains transiently until it is shed or degraded. A microbial virus has a broader niche in the microbiome that depends on the replicative strategy (lytic or temperate). Lytic microviromes exert top-down regulation of microbial populations similar to predation, which maintains microbiome diversity by preventing overgrowth by dominant microbes, reviewed by Nanda et al. ⁹, Henrot & Petit ¹⁰, and others. Temperate microviromes influence microbial competition by increasing horizontal gene transfer between host microbes⁸, which can increase the resilience of stable host microbiomes¹¹ and can increase pathogenicity in unstable microbiomes¹². A microvirome shifts between predominantly lytic or temperate states in response to changes in microbial density, microbial metabolism, and phage coinfection rates^13,14, which has substantial downstream effects on the microbiome. In marine animals, the microbiome is critical to immunity and health, yet the ecological dynamics of the marine animal virome remain understudied.

The animal virome and microvirome exert distinct pressures on animal hosts: animal viruses are primarily associated with transmission between individuals or zoonosis, while microviromes are linked to dysbiosis events, such as coral bleaching and disease associated with bacterial infections. In some cases, animal viruses and the microvirome have compounding detrimental effects on host health by lysing animal cells and beneficial microbial symbionts, respectively¹⁵. The nature of the viral-animal relationship is often determined at the interface between animal and environment, where viruses transition via shedding or colonization, or environmental changes disrupt existing microbial communities. The interaction is most apparent at the epidermal surface, which is a critical immune barrier and ecological interface. The immune role of the epidermal virome is well recognized in terrestrial animals^16,17 but remains underexplored in marine species, despite their long evolutionary history and constant exposure to pathogens. Understanding viral ecology at the animal-environmental interface is key to predicting infection and dysbiosis risks. We present the epidermal ecotone model as a framework to study host microbiomes and highlight the key role of viruses in the complex relationship between marine animals, microbiomes, and the environment.

The Epidermal Ecotone: An Interconnected, Living Micro-Landscape

We propose that the epidermis acts as a selective gradient for viruses between the animal habitat and the water column habitat—a transitional zone that combines components and inhabitants of each adjacent environment and contains a suite of viral inhabitants unique to the transitional space. The epidermal ecosystem is a combination of three main micro-habitats: the integument, the animal tissues just beneath the surface of the skin; the surface, comprised of the external surface of the epidermis and mucus layers produced by marine animals¹⁸; and the aura, the water in direct contact with the animal surface. These three discrete but interconnected microbial habitat zones are reminiscent of an ecotone—an ecologically unique region where multiple ecosystems meet and exchange biota and nutrients¹⁹.

From a microbial perspective, a marine animal is like an island and the epidermal ecosystem is analogous to a microscopic coastline. In a coastal ecosystem, the ocean habitat interfaces with the terrestrial habitat, species are exchanged from land to sea, and vice versa, and there is an intermediate habitat (e.g. saltmarsh, rocky intertidal, estuaries) that hosts species from both land and sea and additional unique sets of species that only exist in the intermediate area. In the epidermal ecosystem of marine animals, the ocean interfaces with the tissue habitat at the epidermal surface, often with a mucosal intermediary layer. Tissues contain viruses adapted to infect the host animal, the aura contains the local environmental viruses, and the surface has a unique community that includes components of both. Just as intermediate coastal habitat structures vary depending on the abiotic and biotic conditions of the land mass (e.g., climate, geology), the structure of the surface habitat depends on the physiology of the host animal’s outer integumentary layer.

The tissue habitat of the epidermal ecotone encompasses the layers of a marine animal’s tissue that interface with the outermost epidermal layer and connect to the internal tissues and circulatory system. The integumentary layer is a critical immune barrier that varies widely among marine animals in thickness and morphology, ranging from a single layer of epithelial cells in cnidarians that is only ~50–100 µm deep^20,21, to chitinous exoskeletons in marine arthropods²², to centimeters-thick stacks of differing cell types in higher vertebrates like whales²³ and dolphins²⁴. The biochemical composition of epidermal tissues also varies across marine animals; for example, the epidermal tissues of soft-bodied invertebrates are often dominated by proteins like collagen²⁵, especially cephalopods²⁶, while crustacean epidermis contains large proportions of the polysaccharide chitin²⁷. Marine animal tissues have unique lipid profiles that vary across the marine food web^28–30, which creates a selective effect in the integumentary tissue related to viral capsid/envelope polarity. Animal viruses must adapt to penetrate the integumentary layer of the host species and infect viable cells, and thus the selective power of the epidermal barrier is key to the role of the tissue habitat in the epidermal ecosystem.

Epidermal surface topography is an important facet of integumentary defense that varies among marine animals (Fig. 1) and structures the epidermal microbiome of some marine animals³¹. Surface morphologies range from smooth, soft, exposed epithelial cells in many cnidarians³² to dense layers of hardened scales in teleost fish³³ and may alternate between both on the same animal, as seen in stingrays³⁴. Cetaceans have hydrodynamic grooves in the outer epidermis²³, invertebrates, such as sea cucumbers have highly papillated epidermis³⁵, and cephalopods deliberately change the rugosity of their skin instantaneously from smooth to rough textures for camouflage³⁶. The rugosity of the external layer determines the flow interactions with the water column³⁷, which influences colonization by environmental microviromes and animal viruses. Rougher micro-scale textures increase hydrophobicity and decrease microbial attachment and biofilm formation. Pelagic shark skin repels environmental microbiota and potentially viruses with a rough hydrodynamic layer of dermal denticles³⁸, which leads to the development of species-specific microbiomes^39–41 and microviromes⁴². The morphology of the epidermal surface can therefore complement the tissue barrier by repelling harmful microbes and viruses and cultivating commensal and beneficial taxa.

Fig. 1. Epidermal surface habitat varies among marine animals.

a–c Electron micrograph of microbes visible on marine animal epidermal surfaces with variable mucosal habitats: a shark with patchy mucus (a, left), a shark with contiguous thin mucus (b, center), and a stingray with thick contiguous mucus with complex structure (c, right).

Mucus is a key habitat feature on the epidermal surface, reducing the burden on the tissue as an immune barrier and aiding the recruitment of a commensal microbiome and microvirome. Mucus provides a nutrient-rich and selective microbial habitat with a complex three-dimensional structure comprised of an intricate network of bonds between numerous glycoproteins, polysaccharides, peptides, lipids, and other biochemicals^43–46. Most marine animals secrete epidermal mucus for purposes including locomotion⁴⁷, parasite avoidance⁴⁸, feeding mechanisms⁴⁹, and reproduction⁵⁰. Mucosal composition is unique to the secreting organism^51,52; the abundance, physical properties, and biochemistry of the mucosal habitat (pH, density, carbohydrate structure, etc.) vary among species and across the body of a single individual^34,52,53. The production and retention of mucus varies among marine animals, which affects viral community structure⁵⁴.

The aura habitat, as it pertains to the epidermal ecotone, consists of the water immediately surrounding the external surfaces of a marine animal⁵⁵. The water around the epidermis acts as both a source of viral colonization from the environment and a receptacle for viruses shed from surface tissue or mucus⁵⁵. The epidermal viromes of marine animals have adapted to the biotic niche through evolution and co-evolution with hosts, forming unique core communities distinct from the water column⁴¹. However, phages and their bacterial hosts persist in diverse environments and proliferate where resources are optimal^11,14,56. The marine environment is the largest reservoir of viral taxa on the planet¹⁶, with upwards of 195,000 genotypically distinct viral populations in global oceans⁵⁷, representing a massive source pool of viruses that interact with the epidermal surfaces. Proximity to the epidermal surfaces of marine animals offers unique combinations of resources and habitats for environmental and epidermal taxa to colonize.

Two key features of epidermal innate immunity are based on the transition of animal viruses between epidermal habitat zones. The first feature is the inhibition of animal viruses entering the tissue from the aura, i.e., preventing a viral infection. The second feature is facilitating the expulsion of animal viruses out of the tissue and into the aura, i.e., shedding to clear a viral infection. Regulating viral transitions between epidermal habitats is thus critical in maintaining animal health, but not all viral habitat transitions are inherently immune-regulated. Animals passively curate a niche-adapted microvirome to regulate the epidermal microbiome and enhance immune resistance to microbial pathogens^11,17. The sloughing of the microvirome and animal viruses from the epidermal surface into the aura is understudied, but represents both a substantial source of viral colonization between animals and an essential layer of animal immunity in the marine environment.

We suggest that the epidermal ecotone mirrors the niche overlap common to landscape ecotones⁵⁸ because of the unique ability of viruses to access and adapt to different environments. Their small size is advantageous for transport across physical barriers⁵⁹, and the capacity of some viruses to infect hosts from disparate environments enables them to spread and establish between distinct ecosystems ⁶⁰. Once a virus has transitioned into a new habitat, it is subject to different selective pressures and ecological forces that will determine replicative success. In the following sections, we will synthesize virome, microvirome, and microbiome attributes in each of the ecotone habitats (tissue, surface, and aura) in marine animals and comparable ecosystems to hypothesize which key factors shape the composition and replicative ecology of viral communities across the epidermal ecotone (Fig. 2).

Fig. 2. Overview of the Epidermal Ecotone.

Conceptual representation the epidermal ecotone, including the relative influences of animals and the environment, the factors shaping community structure and replicative strategies in animal viruses and the microvirome, the mechanisms of viral clearance from the tissues outwards, and the mechanisms of viral colonization from the aura inwards.

The Tissue Virome: An Immune-Regulated Reservoir

The tissue habitat is a direct line to the animal organs, which means the animal's immune system is the key structuring force in determining virome composition and dynamics. Epidermal tissue acts as a physical barrier fortified with additional immune properties that vary broadly across marine animals^52,61,62. However, the fact that the epidermal tissue is nearest to the environment means it is a constant target for infection by animal viruses. Hundreds of animal viruses have been identified among marine animals, including herpesviruses, circoviruses, caliciviruses, poxviruses, and others (reviewed by Bossart & Duignan⁶³, Wellehan & Cortes-Hinojosa ⁶⁴, and Amabalavanan et al. ⁶⁵), many of which present symptoms in the epidermis.

The animal virus component of the tissue habitat includes active viruses, latent viruses, and viral genomic relics. Many animal viruses have developed methods of evading or suppressing the animal immune response, such as antigen alterations, interfering with apoptosis, and signaling disruptors^66,67. The presence of animal viruses in the tissue is not inherently indicative of virulent infection as some animal viruses can diffuse through epithelial cells without active infection⁶⁸. Some animal viruses can also enter a latent state by integrating into the animal genome after penetrating the tissue during virulent outbreaks⁶⁹ or transferring from other infected organs through the bloodstream⁶³. Latent animal viruses remain in an animal's genome until they are triggered by cellular stressors or immune suppression⁷⁰, therefore, physiological stress is a structuring force in the tissue animal virus over time. Integrated viruses spread to the animal’s offspring through vertical transmission⁷¹, and over evolutionary timescales, some integrated viral genomes are mutated to the point where they are no longer expressed, creating genomic relics^72,73. Viruses in the Circoviridae family occur on tissue from healthy individuals of tuna⁷⁴ and coral^15,75, and Herpesvirus species are present in healthy sea turtles⁷⁶ and corals¹⁵, but their origins and whether they are active, latent, or relic viruses remain an outstanding question.

Animal viruses move from cell to cell through various mechanisms, reviewed by Cifuentes-Munoz et al. ⁷⁷, but cellular differences in tissue layers influence viral transport between internal tissues and external tissues. The living and highly metabolically active epithelial cells of fish⁷⁸ and invertebrates⁶² are more conducive to viral transport than the hardened layer of keratinized cells common to the outer epidermis of marine mammals⁷⁹. The tissue habitat is adapted for viral movement outwards but not inwards; marine animals have various mechanisms to clear viral infections from their tissues by shedding or releasing viral particles through epidermal secretions^80–82, lesions or growths^83,84, and excrement^85,86. However, some animal viruses have adapted to convert immune clearance mechanisms into methods of spreading to new hosts, exploiting actin-mediated secretion pathways⁸⁷ in epithelial and goblet cells^44,52 and facilitating viral transport from internal tissues to the epidermal surface for release into the aura.

The microvirome of the tissue habitat relies on passive colonization from other parts of the body. Approximately 31 billion phages breach the mammalian gut to enter the bloodstream every day⁸⁸, which means epidermal tissues with blood vessels may contain some components of the gut microvirome. Although it is currently only described in mammalian cells (mostly human and mouse models), we predict that similar activities occur in the epidermis of marine animals. The uptake of phages into tissue depends on host cell type and phage morphology, with size selective mechanisms varying between epithelial cell types⁵⁹. Given the constant abundance and large numbers of phages in the marine environment, it is possible that some environmental phages can enter the tissues of marine animals, especially soft-bodied invertebrates with more permeable epidermis. The success rate of colonization would depend on the animal’s immune response to phages, which ranges from negligible to inflammatory in fish⁸⁹ and mammals⁹⁰. Tissue microviromes are a valuable part of the coral epidermal ecotone, especially the viruses of symbiotic intracellular bacteria and algae^15,91, but the role of the microvirome in other marine animal tissues is unresolved.

The community structure of the tissue microvirome depends on the level of partitioning between the host animal and its microbiome, which varies across animal phylogeny. As the complexity of an animal’s adaptive immune system increases, the degree to which microbiomes and microviromes are partitioned away from the host tissue increases⁹². Invertebrates, such as corals⁹³, nudibranchs⁹⁴, and sea jellies⁹⁵ sequester microbes inside epithelial cells for photosynthetic symbioses, populating the tissue microvirome with phages and microalgal viruses. Animals with more complex immunity, such as squid and other deep-sea animals sequester symbiotic microbes inside specialized organs, and microbes heavily modulate the functions of the animal cells during development^96,97. Higher vertebrates, such as mammals and fish, compartmentalize microbes into specific microbe-rich organs with heavy immune regulation like the gut^98,99, but enforcing barriers to microvirome movement is an immune challenge in the case of phages that diffuse through the epithelium.

The adaptive immune system of marine animals has been contending with viruses for ~ 500 million years⁶² and is constantly diversifying defense mechanisms. Although the emphasis of epidermal-viral interaction is on simple host-infection systems, the relationship is more complex and provides opportunities for research and therapies. An increasing number of marine animals are suffering epidermal injury from marine debris¹⁰⁰ and chemical interactions¹⁰¹, which heightens the exposure of epidermal tissue to environmental animal viruses and pathogenic environmental microbes that could be susceptible to the epidermal microvirome. The exploitation of marine bacteriophages for phage therapy to reduce infections and antibiotic resistance in aquaculture¹⁰² and human medicine¹⁰³ is promising and currently being investigated. The viability of phage therapies for marine animal injuries is dependent on the immune response of the tissue habitat, which has not been described comprehensively for many species.

The Surface Virome: a Chemically Dynamic Conveyor Belt

The surface of a marine animal is a dynamic and selective environment for microbial and viral colonization. The exact mechanisms by which marine animals first acquire their surface virome are still unknown for most species, but we predict there are contributions of taxa from both the tissue and the aura, which are shaped by morphological and physiological factors over the animal’s life to form a unique surface community. Animal viruses are present on the epidermal surface, but the surface virome is generally dominated by the microvirome adapted to the mucus layer^42,74,104. Common taxa include tailed phages from the families formerly known as Myoviridae, Podoviridae, and Siphoviridae^42,74,105, which have been reclassified into different families, such as Herelleviridae and Autographiviridae. Viruses that persist in this niche must therefore be compatible with both the physical and biochemical conditions of the epidermis and mucus¹⁰⁶, and the microvirome requires microbial hosts that are also biochemically adapted to the surface habitat. The principal forces shaping the composition and replicative strategies of the surface virome are morphology, physical disturbance from epidermal and mucosal shedding, and biochemical variability within the mucus.

Epidermal and mucosal shedding is a widespread phenomenon in marine animals. The sloughing of skin cells and mucus acts as a mechanical force that resets or reshapes microbial and viral assemblages^107,108. Different animals shed the surface layer at seasonal¹⁰⁹, monthly¹¹⁰, weekly¹¹¹, or even continuous^112,113 timescales. Increased mucosal shedding is a common means of clearing animal viruses from the tissues¹¹⁴. While shedding is a useful immune tool for clearing viral infections from an individual animal, it increases animal virus infectivity in an animal population by releasing infected epidermal tissue into the environment^82,114. Additionally, shedding mechanisms that alter the chemical components of the skin, like the molting process in crustaceans, heighten susceptibility to infection by animal viruses while the new exoskeleton hardens^115,116. Similarly, regular mucosal shedding maintains microbiome stability in coral mucus, but excessive shedding increases vulnerability to pathogenic infection¹¹⁷.

Mucosal shedding adds an extra buffer against animal viruses before they reach animal tissues and helps to structure the epidermal microvirome, especially the phage community. Mucus turnover disperses mucosal microbes and their viruses into the water column¹¹⁸, restricting opportunities for long-term microvirome colonization to the taxa best adapted to surface niches. For example, some phages are “ambush predators” that rely on immunoglobulin-like domains to adhere to mucosal glycoproteins to increase encounter rates with host microbes¹⁷, while others are “pursuit predators” that utilize tail spike depolymerase proteins to cleave mucosal polysaccharides and move more quickly through the mucosal habitat¹¹⁹. For most phages, diffusion through mucus becomes limited at high glycoprotein concentrations¹²⁰, thus the outermost layers of mucus are typically sloughed off before maladapted phage colonizers from the environmental microbiome penetrate deeper or into more stable regions. Adaptation to the chemical conditions of the mucosal environment is, therefore, key to microvirome persistence.

Fluctuations in mucosal chemistry influence the microvirome by modulating viral infectivity and increasing microbial susceptibility to viral infection through cellular stressors. Mucosal microviromes become lytic when host microbes are chemically stressed¹¹, creating temporal heterogeneity that affects microvirome community structure. Dietary shifts alter the metabolite content of epidermal secretions^29,121, with changes in ingested nitrogen and carbon altering the surface mucus within 24 hours¹²². Food deprivation, conversely, leads to declines in glucose levels¹²³, adding nutritive stress to microbes that metabolize monosaccharides. Osmotic stress increases soluble proteins and lactate levels in fish mucus¹²⁴, and air exposure reduces glucose and elevates cortisol in fish mucus¹²³. Thermal stress induces production of stress-related biochemicals, such as cortisol, malondialdehyde, and elevated mucus protein and lipid content in fishes and corals^125–127. Pathogenic microbial infections alter mucosal protein expression; Aeromonas hydrophila infection in fish changes the expression of over 100 different mucosal proteins¹²⁸. Antimicrobial compounds produced and secreted by animals or mucosal microbes are common agents of prophage induction. Crustaceans preemptively secrete antimicrobial peptides to reduce the abundance of certain bacterial taxa during molting^108,129. In stingrays and skates, isolated mucus-associated bacteria secreted at least one antibiotic, many of which were broad-spectrum¹³⁰ and thus could cause shifts in the microvirome by inducing phages across diverse microbial taxa. Stressors that change the chemical landscape of the mucus have downstream effects on the microbiome, which we predict to increase prophage and provirus induction in the microvirome.

The surface habitat is an environment defined by periodic disturbances, both physical and biochemical. Periodic disturbance is hypothesized to preserve diversity and resilience in an ecosystem^131,132, and we contend that epidermal and mucosal shedding are analogous to habitat disturbances that work in concert with the microvirome to preserve diversity and resilience in the epidermal microbiome. Excessive turnover of surface tissues and mucus leaves the underlying dermal tissues exposed to animal viruses. Lack of habitat turnover allows for overgrowth of dominant taxa and increases the potential for virulence in the microbiome¹³³. Investigating the interplay between biochemistry and the microvirome dynamics of the epidermal surface habitat will provide useful insight into the mechanisms governing the first line of the animal immune defense against the environment.

The Aura Virome: a Viral Gateway

The aura habitat is the portion of the water column that physically interacts with the animal surface, the area in which viral taxa are exchanged between the environment and the surface habitat. The aura virome is therefore a variable mixture of local marine environmental viruses and the viral taxa the animal has shed from the surface habitat. The line between the environment and the aura is difficult to define- the precise “relevant” distance from the epidermis is subjective and is influenced by animal mobility and the fluid dynamics of the micro-currents surrounding the animal.

The potential diversity of viruses contributed from the seawater to the aura is substantial; the marine virome has upwards of ~ 120,000 predicted genotypes¹⁶, and there are an estimated 5000 different viral genotypes in a given 200-liter volume of seawater⁵⁶. The composition of the marine virome follows strong biogeographical patterns^{56,57,134,135}, and is sensitive to fluctuations in environmental variables, such as salinity¹³⁶, temperature and chemistry¹³⁷, and UV radiation¹³⁸, therefore the composition of the marine virome follows seasonal and temporal trends^139,140. Depth and seasonal variation have interactive effects on taxonomic composition of the marine microvirome due to temperature-driven stratification of the water column^141,142. The water column is a major source of animal viruses and microvirome taxa contributed to the aura virome, and therefore, we expect the environmental taxa contributed to the aura virome to be guided by the same environmental parameters.

Animals are major contributors to the aura, as viruses are passively expelled into the environment when animals release cells and parts of the mucus layer into their surroundings¹¹⁸. Free-floating biofilms and mucous-like substrates are common in the marine environment, including strands of mucus or other labile compounds secreted by animals^55,143 and algae^144,145. Microbial communities generate planktonic mucosal habitats by producing biofilms on marine particulate matter¹⁴⁶ or microplastics^147,148, thus the presence of adequate substrate to support microbial hosts will enable the microvirome to persist in the aura. The composition and replicative ecology of the aura virome are governed by the same abiotic environmental variables as the water column virome, but the animal influences the aura habitat through shedding and physical movements. The degree to which animals influence the aura is yet to be determined.

Mobile marine animals have hydrodynamic body plans to reduce drag while swimming and maximize their energetic efficiency¹⁴⁹, and the same properties that reduce drag also make it more difficult for microbes and viruses in the aura to colonize the epidermal surface³⁸. Different animals experience different water movement over the epidermal habitat depending on body shape¹⁵⁰ and epidermal morphology¹⁵¹, which affects the depth of the ‘no-slip’ boundary layer of laminar flow across their body and thereby determines their relationship to the aura virome. Laminar flow parallel to the epidermis increases encounter rates between viruses and the epidermal surface, but the microscale texture of the epidermis influences recruitment success. For example, pelagic sharks are in constant motion and repel environmental microbial taxa from their epidermal surface with hydrodynamic epidermal morphology, minimizing colonization potential from the aura virome³⁸. Animals exhibit differences in the proportions of laminar and turbulent flow surrounding their bodies as they swim^152,153, which we predict to influence the rates at which they collect and shed animal viruses and microvirome.

The diversity of viral taxa a mobile animal will encounter through the aura depends on the animal’s spatial usage. Marine animal species migrate vertically hundreds of meters through the water column. The water column virome is stratified by depth^{141,154–156}, and therefore animals will be exposed to broad variation in viral communities during vertical movements. Billions of zooplankton in every ocean migrate daily between the surface and the deep sea¹⁵⁷, drawing their predators to the ocean depths¹⁵⁸. Sperm whales often feed on the sea floor below 500 m¹⁵⁸, and large sharks move from the surface to below 150 m depths, including tiger sharks¹⁵⁹, white sharks¹⁶⁰, and whale sharks¹⁶¹. Sudden change in temperature, salinity, pressure, or UV exposure induces some prophages¹⁶², therefore an animal diving thousands of meters in a short time and returning to the surface may shock components of the microbiome and trigger prophage induction. To the best of our knowledge, the effect of deep dives on animal microviromes has not been investigated, but could affect viral mixing and oceanic carbon fluxes.

Stationary marine animals have a more stable relationship with the aura virome than migratory and mobile species. Benthic invertebrates like corals and sponges constantly exude portions of their microbiome as they slough their mucus layer, to such a degree that there is a distinct microbial aura that surrounds coral reefs at a macro-ecosystem level^55,163. The rate of exudation varies among individuals in the same area over time, depending on the species and environmental conditions^43,112. For example, corals increase mucus shedding when exposed to air at low tide¹⁶⁴, at increased light levels¹⁶⁵, and higher temperatures¹⁶⁵, therefore the aura surrounding benthic animals near the ocean surface is likely to contain more mucosal taxa than auras surrounding deeper species. Hydrodynamics are also relevant to the contributions of benthic animals to the aura virome¹⁶⁶, as water mixing introduces and removes taxa from surrounding environments. The overall microvirome flux between benthic organisms in reefs and the aura has not yet been quantified, but has major implications for ecosystem health.

The microbialization of coral reefs is a case study of the value of aura virome dynamics in ecosystem health. The aura microvirome helps to regulate microbial populations in healthy reefs through viral lysis, while on algae-dominated tropical reefs the microbial populations overgrow and the microvirome shifts toward temperate strategies^167,168. The microvirome dynamics of the aura are responding to changes in the metabolism of the aura microbiome as the community feeds on the more labile carbon secreted by algae¹⁶⁷. The result for the environment is decreased oxygen levels due to microbial respiration¹⁶⁹, and the result for the animal habitat is increased exposure to pathogens enhanced by virulent prophage elements^133,167,170. The impacts of viral ecology in the aura habitat are already manifesting on coral reefs, but further study is required to understand the full extent of the aura virome’s role in the health of marine ecosystems.

The Value of the Epidermal Ecotone as a Study System

The epidermal ecotone is still a new frontier in the viromics field. There is growing interest in marine viromics and host-associated viromics separately¹⁷¹, but marine host-associated viromics are rarely included in broad-scale studies. The epidermal ecotone is a viable model system for studying the tripartite animal-microbe-virus relationship. The mucosal interactions and relationships to diet- and stress-based fluctuations in the surface microvirome may provide foundational knowledge that improves our understanding of similar processes in other animal-associated microbial systems like the gut or respiratory tracts, which are often less accessible due to ethical regulations⁹⁹. Studying epidermal ecotones provides a minimally invasive way to investigate microbial and viral interactions, which are translatable to other systems. In the following section, we will provide examples of open ecological questions well-suited for study in the epidermal ecotone.

Spatiotemporal Variability in Viral Communities

Ecotones at the landscape scale are dynamic and spatially heterogeneous patchworks of habitats rather than uniform blocks of adjacent habitats with clear lines¹⁷², and these non-contiguous habitats have unique ecological properties and community dynamics^173,174. Our review shows that the same would be true of the epidermal ecotone: skin texture, mucus thickness and composition, and water column parameters are not consistent across the body of a marine animal. Small differences in the habitat may create certain areas across the body that are more vulnerable or resistant to infection. Therefore, sampling marine animal viromes across spatial and temporal scales is valuable.

Epidermal microbiomes exhibit biogeographical patterns, and we predict the microvirome to behave the same way. On the individual organismal scale, epidermal microbiomes exhibit taxonomic and functional differences across the body^175,176. At the geographical scale, animal microbiomes are more similar between animals within an aggregation compared to aggregations from different global regions¹⁷⁷ and across different sites on the same coastline⁴¹. The viral abundance in coral mucus is on average ~ 15- to 20-fold higher than surrounding water¹⁷⁸, and we suggest that mucosal surfaces of other animals may harbor high numbers of viruses.

The boom-and-bust cycle of viral replication and persistent cell and mucus sloughing that occurs in the epidermal ecotone implies a stochastic viral community; the rates of community fluctuation in the epidermal virome are unknown but are likely to change substantially over short time scales. In the marine environment, viral abundance fluctuates by an order of magnitude over the course of a day¹⁷⁹. Fish skin microbiome taxonomy changes in response to environmental salinity change in brackish water on a scale of days¹⁸⁰, and microbiomes in different fish body areas (i.e., gills, skin surface) fluctuate on the scale of weeks¹⁸¹. The epidermal microbiome of an aggregation of leopard sharks showed taxonomic fluctuations over multiple years¹⁸². The long-term stability of the microvirome is ultimately dependent on the long-term stability of the host supply in the microbiome. Temporal stability or variability in the virome will affect the community resilience of the epidermal ecotone, and thereby affect animal health; it is an important piece of the immune puzzle that warrants examination, beginning with the establishment of temporally stable core taxa.

Ecosystem-Scale Microbiome Connectivity

Animals directly exchange components of the epidermal virome within species and between species via physical contact. Pelagic fish scrape their skin against the rougher skin of sharks to remove parasites¹⁸³, and the transfer of the mucosal virome is inevitable. Interactions between sessile animals (animals stuck to a hard substrate, e.g., corals, anemones, and sponges) and motile animals (animals that swim through the water) increase the dispersal potential of the virome. Dolphins rub their skin against specific coral and sponge species, which has been hypothesized to treat or prevent skin infections using antimicrobials in the coral mucus¹⁸⁴. Clownfish skin mucus microbiomes converge with their symbiotic anemones through a 10-fold increase in Cellulophaga spp¹⁸⁵. We therefore hypothesize that there is a distinct, albeit difficult to quantify, portion of animal-driven connectivity in the global virome beyond simple pathogenesis which has yet to be examined.

Animal Resilience to Global Climate Change

Rising ocean temperatures will affect the microvirome and animal virus components across the epidermal ecotone. Environmental stressors increase susceptibility to viral infections, and climate change is hypothesized to increase the global prevalence of marine animal viral diseases, especially strains with high thermal tolerance¹⁸⁶. In fish, elevated temperature increases immune function^147,148, but the replication of certain thermally adapted viruses, such as infectious hematopoietic necrosis virus (IHNV) can outpace the fish immune response at higher temperatures¹⁸⁷. Thermal stress also induces latent animal virus production, as seen in Herpesviruses in corals during marine heatwaves¹⁸⁸. Temperature influences bacterial metabolisms, which are a major driver of microvirome lytic productivity¹⁴. In warmer months, bacterial metabolisms are higher and therefore support higher phage reproduction¹⁸⁹, while cooler temperatures shift communities toward lysogeny^190,191. Increasing global temperatures are hypothesized to increase marine bacterial metabolic rates¹⁹² and affect marine phage ecology by altering the dynamics of lytic/lysogenic cycles¹⁴. Increases in viral production from both the environment and the host will create a higher viral load in the aura habitat.

Anthropogenic stressors, such as pollution and habitat loss, manifest in the animal virome, weaken the immune systems of marine animals, and increase susceptibility to viral infection^193,194. High levels of river or wastewater treatment runoff may introduce human or agricultural microbes and viruses to marine animals¹⁹⁵. Human chemicals and pollutants influence lysogeny in the virome; trace concentrations of industrial pollutants¹⁹⁶, sunscreen¹⁹⁷, and heavy metals¹⁹⁸ induce lysis in temperate marine phages. The response of marine animal epidermal viromes to pollutants and other chemical stressors remains an outstanding question but may have substantial implications for conserving sensitive species.

The Role of the Virome in Animal Health and Immunity

The epidermal ecotone harbors complex interaction networks between the microvirome and animal viruses, with profound impacts on animal health. For example, viral mortality of symbiotic dinoflagellates leads to viral infection in thermally stressed corals during bleaching events^15,199. Tissue mortality caused by viruses in the epidermal habitat triggers shifts in microbial populations²⁰⁰, altering the microvirome. A sudden, drastic shift in a previously stable mucus microbiome, known as dysbiosis, leaves the animal more vulnerable to pathogens^201,202. Host microbiomes on average are 5x less responsive to environmental change than seawater²⁰³, however, the epidermal ecotone buffer may be lessened by the compounding effects from climate change and biotic interactions. The stability of the epidermal microbiome and virome—and therefore the health of the animal host—hinges on the resilience of the epidermal ecotone.

Epidermal injuries are common among marine animals, and the mucosal microbiome is an integral part of the immune response in wound healing. The mucus acts as both a physical barrier to pathogen invasion and a habitat for endogenous commensal microbes and phages that outcompete and eliminate environmental pathogens^2,11,204. Coral mucus harbors reservoirs of phages that infect pathogenic Vibrio species and induce phage lysis during Vibrio outbreaks²⁰⁵. Mucus production increases in response to epidermal injury in fish to widen the barrier between environmental pathogens and the injured tissues and concentrate useful mucosal compounds near the injury^52,206. Dozens of antimicrobial compounds have been isolated from the mucus of fish^207,208, sea slugs²⁰⁹, and crustaceans²¹⁰, many of which are useful in fighting human infections.

Epidermal wound infections are characterized by reduced microbial diversity as pathogenic strains proliferate²¹¹, which increases encounter rates with commensal phages that infect the growing bacterial populations²¹². There is an energetic tradeoff for bacteria between harboring genes for resistance to diverse antimicrobials and defense against phages^213,214, thus the necessary resistance profile for remaining in the mucosal habitat opens a window for commensal phages to infect. Phages have flexible genomes and evolve rapidly^215,216, which increases the energetic cost for microbes to evolve phage resistance²¹⁷. We propose that the marine animal immune system and the mucosal phage community work in concert to prevent bacterial infection of the epidermis.

Establishing Baseline Data for the Epidermal Ecotone

Establishing baselines is critical for predicting and understanding future shifts in viral ecology, but there is currently little data on the composition and abundance of the marine epidermal virome unrelated to disease states. The value of applying macro-ecological perspectives to microbiomes is recognized^218,219, and this review illustrates the applications of ecological frameworks in the epidermal ecotone. Earlier sections highlighted knowledge gaps in ecological mechanisms for each habitat in the ecotone, from interactions between the animal immune system and the microvirome to the exchange of viral taxa between animals and the environment. Filling these knowledge gaps requires specialized methodology and an increased sampling effort of marine animal viromes across animal species, spatial scales, and temporal scales. Our review demonstrates the complexity and fluidity of the epidermal ecotone system that must be considered when examining marine animal viromes.

Sampling each habitat of the epidermal ecotone separately requires a precise experimental approach. Tissue viruses should be isolated from extracted tissue after washing the sample to remove any contamination from the surface mucosa²²⁰. Mucosal virus samples can be taken passively from mucus removed through “milking”²²¹ or collected from the skin out of the water to reduce environmental contamination³⁴. The aura should be sampled in situ by directly sampling the water interacting with the epidermis, rather than the entire water column⁵⁵. Surface swabbing is a common, quick, and affordable method for surface microbiome collection, but it has drawbacks for sampling the epidermal viromes of marine animals. The chemical interactions between the fibers in the swab and the viral capsid are affected by the swab material²²². Applying buffers to the swabs enhances sample preservation^223,224, but further affects the binding chemistry of microbes and viruses²²². Swab efficacy is influenced by the texture of the animal surface²²⁵, therefore the variation in animal epidermal surface morphology warrants consideration when sampling.

A novel method has been developed to collect microbiomes and microviromes from marine animal epidermal ecotones using blunt recapture syringes. This technique has been used on a wide range of species, from sharks^39,40,42,182 and rays⁴¹ to corals²²⁶. The syringe is filled with sterile seawater produced from a tangential flow filter¹⁶³, which maintains the tonicity of the epidermal ecosystem to avoid cellular lysis and the blunt fitting on the end minimizes epidermal abrasion. The sterile seawater is flushed over the epidermis as the plunger is depressed, dislodging the mucus, microbiome, and virome from the epidermal surface with a combination of rinsing and suction, then the microbial slurry is recollected in the reverse side of the syringe. This technique can be performed underwater in situ to reduce animal handling stress from air exposure or fishing¹⁷⁷ and minimizes physical abrasion to prevent contamination from blood and epidermal cells²²⁷. The combination of rinsing and suction removes the mucosa and viruses attached to the epidermis, thereby collecting a simultaneous subsample of the entire epidermal ecotone. The viral fraction is separated from the microbial fraction via size separation and enrichment for metagenomic sequencing⁴².

Isolating and culturing viruses from marine animal epidermal ecotone is progressing, and uncultured viromics allow for high-throughput analyses from in situ samples^228,229. The gaps are numerous in the global viral dataset²³⁰, which makes it difficult to identify viral taxa on marine animal epidermis beyond the order and family level⁴². The recent reclassification of the former Caudovirales order of phages into numerous other groups warrants the reanalysis and re-annotation of existing datasets, which would likely increase the resolution of microvirome taxonomy comparisons across epidermal ecotone datasets. Genome assembly provides a means for analyzing viral diversity, as contiguous genome sequences (contigs) can be generated without taxonomic assignment and used in a manner akin to comparing ASVs in metabarcoding analysis. The relatively small genome size of most viruses (from as small as ~ 4,000 base pairs²³¹ up to ~ 700,000 base pairs in some cases²³² but generally between 25,000 – 200,000 base pairs²³³) allows for reference-independent comparison of community level composition with better resolution than comparable techniques for bacterial datasets with larger genome sizes. Methodology for resolving viral metagenomic datasets computationally has made enormous strides in recent years with the creation of specialized bioinformatic tools designed to eliminate contamination²³⁴, identify reproductive strategies^235,236, predict microbial hosts²³⁷, assemble genomes²³⁸, resolve assembled viral genomes²³⁹ and annotate genes^240–243 in viral metagenomes. Further sampling of epidermal ecotones across more animal species and geographic locations and environments will improve the databases and reduce the skew towards human-associated taxa.

Conclusion

The marine animal epidermal ecosystem is effectively a microscopic viral ecotone comprised of three distinct neighboring habitats that interact to form diverse and dynamic viral communities key to animal health. The core viral communities are shaped by the coevolution between animals and their viruses, the physical and biochemical properties of the epidermis and mucus, the dynamics of the microbiome and microvirome, and environmental fluctuations. The variables governing viral dynamics in the epidermal ecotone will vary among species, within individuals, across spatial scales, and through time. Increased sampling effort across a broader range of marine animals and improved techniques are necessary to understand the complex ecology of epidermal viromes. The epidermal ecotone concept provides a foundation for exploring the intricacies of viral dynamics on marine animals and the role of commensal viruses in organismal health.

Author contributions

RDH conceived of, wrote, and edited the manuscript. EAD wrote and edited the manuscript. All authors have read and approved this edition of the manuscript.

Data availability

No datasets were generated or analyzed during the current study.

Competing interests

The authors declare no competing interests.