Introduction

Animal microbiomes influence host physiology, behavior, and evolution, yet have been studied sparingly in most fishes, including elasmobranchs (sharks, skates and rays). Understanding elasmobranch microbiomes is emerging as a research priority given the biological and ecological significance of this major vertebrate lineage. Representing over 1130 species, elasmobranchs occur in marine and freshwater habitats across the globe [1]. As carnivores, elasmobranchs shape food webs and move large amounts of carbon and energy through diverse feeding modes. While most elasmobranchs are generalist predators and feed intermittently, others such as the whale shark (Rhincodon typus) or basking shark (Cetorhinus maximus) are filter feeders, with diets more akin to those of baleen whales (suborder Mysticeti). Despite their diversity and ecological significance, nearly 50% of elasmobranch species are listed as “data deficient” by the International Union for the Conservation of Nature (IUCN) Red List, meaning that information is missing to fully assess their status [2]. For these taxa, we lack basic information on life history, physiology, and inter-species interactions, including those with microorganisms.

Elasmobranchs have traits that suggest unique interactions with microbes. Diverse bacteria are regularly cultured from the blood of healthy individuals [3], raising the question of why these microbes do not trigger an immune response. Indeed, while natural mortality events are rarely investigated and diagnosing elasmobranch disease remains challenging, elasmobranchs appear to be relatively disease-free [4]. Documented cases of cancer in elasmobranchs are exceedingly rare. Further, elasmobranchs rarely experience infections from injuries and appear to recover quickly in the presence of wounds [4,5,6]. Unlike most vertebrates, elasmobranchs naturally synthesize small single chain antibodies that help counteract a broad range of pathogens [7, 8]. Distinctive elasmobranch compounds are being studied for the treatment of certain cancers, age-related macular degeneration, viral infections, autoimmune diseases, and Parkinson's disease [4]. While studies from other systems confirm that microbiomes exert critical effects on animal immune status and health [9, 10], it remains unknown how the immune properties of elasmobranchs interact with or are shaped by the resident microbiome.

Interest in interactions between fish and commensal microbes has increased notably in recent years, although much of this work remains focused on teleost fishes [e.g., 11, 12]. Early work on elasmobranch-associated microbes focused primarily on disease [13] and typically used culture-based approaches to identify a subset of microbial taxa common to elasmobranchs [5, 14,15,16,17,18,19,20]. Only recently have DNA sequencing-based studies begun to provide a holistic understanding of elasmobranch microbiology [10, 21]. These and similar studies are facilitated by sustained efforts to find, track, and sample elasmobranchs in the wild, which can be challenging. Specialized vessels or equipment for sampling elasmobranchs safely and humanely, in addition to research on animals under managed care, have allowed for improved access to individuals (Figs. 1, 2, 3). Such work is critical as it informs our understanding of elasmobranch immunity, disease, and the potential for microbe–host relationships to change under environmental disturbance or managed care.

Fig. 1
figure 1

Sampling elasmobranch microbiomes poses physical and technical challenges. Sampling techniques vary among species, locations, and research groups. Microbiome samples have been collected by freediving and swabbing free-swimming animals (A) or immobilizing individuals out of water and collecting microbial biomass by swabbing or using custom equipment, such as modified suction devices (B with inset). Sampling large pelagic individuals may involve modified vessels equipped with platforms that raise and secure caught individuals (C, D), providing a unique opportunity to sample species that are hard to capture and restrain. Panel A Gill swab from a free-swimming whale shark (Simon Pierce, Marine Megafauna Foundation). Panel B Supersucker sampling device (inset: Michael Doane, Flinders University) being used to sample a leopard shark (Elizabeth Dinsdale, Flinders University). Panel C White shark on submerged OCEARCH platform (Robert Snow, OCEARCH). Panel D White shark on raised OCEACH platform being secured prior to sampling (Robert Snow, OCEARCH)

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Fig. 2
figure 2

Managed care of elasmobranchs in aquariums provides a unique opportunity for sampling microbiomes over time and relative to monitored host and environmental parameters. Exhibits such as Georgia Aquarium’s Ocean Voyager (A) and Sharks: Predators of the Deep (B) are enabling studies to understand the drivers of microbiome structure and its role in host health. Panel A Whale shark swimming in Georgia Aquarium’s Ocean Voyager exhibit (Chris Duncan, Georgia Aquarium). Panel B Hammerhead shark swimming in Georgia Aquarium’s Sharks: Predators of the Deep exhibit (Chris Duncan, Georgia Aquarium)

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Fig. 3
figure 3

Despite the difficulty of sample collection, elasmobranch microbiomes have been sampled from diverse body niches. Swabbing of the skin/mucus (A, E) and gill (B) is relatively non-invasive and captures microbiomes reflecting both host-specific taxonomic signatures, as well as signatures of the surrounding seawater water microbiome. Host-specific signatures may be driven partly by variation in mucus content and prevalence, such as between sharks and rays. Sampling of gastrointestinal microbiomes has involved opportunistic sampling of feces (C) or swabbing of the cloaca (D), with cloacal communities representing a transition between external and internal microbiomes. Few studies have examined microbiome variation along the GI tract in dissected individuals. Diet, intestinal anatomy, and host foraging ecology may influence GI microbiome structure. Panel A Dorsal skin swab of a tiger shark (Mote Marine Laboratory). Panel B Gill swab of a spotted eagle ray (Mote Marine Laboratory). Panel C Aerial photograph of a whale shark defecating (Tiffany Klein, Ningaloo Aviation). Panel D Cloaca swab of a tiger shark (Mote Marine Laboratory). Panel E Dorsal swab of a spotted eagle ray (Mote Marine Laboratory)

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Elasmobranch microbiome research has targeted a small fraction of host species, suggesting that our knowledge of the diversity and function of associated microbes is sparse. We have, for example, a limited understanding of the extent to which microbiome members are shared across hosts and environments and the mechanisms through which microbes interact with the unique physiology of elasmobranchs. To help close this knowledge gap and guide future research, this review summarizes current knowledge of elasmobranch microbiomes based on data from 43 elasmobranch species across 26 studies. Using these important studies as a baseline, we highlight key questions for exploring the roles of microbes in elasmobranch health, physiology, and ecology. We organize the review into subsections covering different niches of elasmobranch anatomy, beginning with the gastrointestinal (GI) niche followed by those of the oral cavity, skin/mucus, and blood (Figs. 3, 4). While microbial pathogenesis in elasmobranchs is not covered in detail in this review, the question of how a commensal elasmobranch microbiome interacts with pathogens is an important target for future research. We direct readers to Garner [22], Borucinska [23], Stidworthy et al. [24], and Stedman and Garner [25] for reviews of elasmobranch pathogens.

Fig. 4
figure 4

Microbiomes differ among elasmobranch body niches. (Marc Dando, Wildlife Illustrator)

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Gastrointestinal microbiomes

Microbes in the vertebrate GI tract affect host digestion, development, immunomodulation, suppression of pathogens, and overall health [26,27,28]. Knowledge of the diversity and function of GI microbiomes is based primarily on mammals, which account for < 10% of vertebrate diversity [29]. However, GI microbiomes are presumed to play similarly important roles in fishes [12]. As in mammals, GI microbiomes in fishes vary among host species [30, 31], individuals [32], life stages [33], locations in the GI tract [34,16, 17, 45]. Many Vibrios exhibit urease activity, raising the hypothesis that urea exchange may influence the gill microbiome and, conversely, that microbiome urease activity may contribute to ammonia production on the gills [61]. Focusing on teleost fishes, Pratte et al. [30] found that the gill microbiome is distinct from other external body sites (skin). Similar culture-independent studies, for example using metagenomics, could help identify metabolic functions enriched in gill-associated microbes compared to those from skin sites less influenced by host nitrogen cycling.

Blood-associated microbes

For vertebrates, it is assumed that having bacteria in the blood is linked to negative health outcomes. While bacteria may enter the blood of healthy individuals, these events are short-lived if the immune system is not compromised. If the immune system is overwhelmed, proliferating bacteria can result in sepsis, a life-threatening organ dysfunction caused by aberrant host response to infection. In contrast to this assumption, bacteria have been cultured repeatedly from the blood of healthy elasmobranchs (Fig. 4; Additional file 1: Table S4). These include Gram-positive and -negative heterotrophs commonly recovered from both planktonic and host-associated marine microbiomes, notably genera of the ubiquitous order Pseudomonales (e.g., Vibrio, Photobacterium, Aeromonas, Moraxella; [16, 18]). A study of 195 individuals representing 12 species recovered culturable bacteria from 21% of sharks and 50% of rays, noting that cultures were more often recovered from pelagic species (38.7%) compared to sedentary species (18.3%) [3]. However, the authors acknowledge that some samples may have been contaminated from needle passage through muscle or skin tissue. Tao et al. [120] also isolated bacteria, primarily Vibrio species, from blood of the lesser electric ray (Narcine bancroftii), with many of these isolates being distinct from reference strains and potentially representing new species of Vibrio, Amphritea, Shewanella, and Tenacibaculum. As many of these genera are also found in marine sediments, the authors posited that microbes may enter the host by ingestion of sediments during benthic feeding. If so, the bacteria would then enter the bloodstream, presumably via entry across the intestinal lining.

The repeated detection of bacteria in elasmobranch blood suggests that non-sterile blood is a baseline condition in this major aquatic group, challenging the classical assumption that bacteria in blood indicates disease. Elasmobranchs are an ancient vertebrate lineage and one of the first to evolve adaptive immunity [121], and therefore, sharks have long been important targets for immunology research [122]. Their immune systems share important properties with those of humans, while also showing key differences, including the presence of rare single chain antibodies [123]. Further, sharks rarely experience infections [4]. If these and other unique immune properties explain, or can be explained by, the persistence of microbes in the blood (outside of a disease state), then characterizing these microbes may have implications for understanding why immune systems evolved differently among vertebrate groups. However, additional work is needed to confirm that bacteria persist as metabolically active ‘residents’ in elasmobranch blood.

Conclusions

Elasmobranch microbiome research has intensified dramatically in recent years. This work has been motivated in part by a need to better understand the health of rays and sharks as these ecologically important animals continue to face significant environmental and anthropogenic stressors [124]. Additionally, understanding of baselines in the microbiome community will allow best care practices for elasmobranch in managed care facilities. Further, the unique physiology of elasmobranchs pertaining to metabolism, osmoregulation, and immunity suggests the potential that elasmobranch–microbe interactions are distinct from those in other vertebrates, including teleost fishes. In cases where poor host health may involve a microbial component—either a specific pathogen or an imbalance in the microbiome (dysbiosis)—it may be unclear if negative health effects are due to resident microbes that changed from commensal to harmful as conditions changed, colonization by outside pathogens, or both. Distinguishing among these processes is a priority but requires a clearer understanding of which microorganisms do or do not constitute health threats in elasmobranchs, as well as studies that assess the microbiome over changes in host health, e.g., due to stress, disease, or wounding and recovery. Such studies remain rare for elasmobranchs, potentially due in part to the relative novelty of considering disease in the context of microbe–microbe interactions [21], but likely also to the challenges of working with these animals.

Sampling elasmobranch microbiomes can be difficult. Not only are many elasmobranchs challenging to capture, but substantial resources are also required to obtain the sample size necessary for statistical analysis. Capturing elasmobranchs can require specialized vessels and equipment to minimize risk to the animals and the researchers. Once captured, live animals must be handled with care and usually only for short periods of time to avoid stressing or injuring the animal. Microbiome sampling may therefore be restricted to quick, non-invasive swabs of the skin or other external surfaces. Elasmobranch fecal samples may be collected only opportunistically and are particularly rare for large migratory or deep-sea species. Fortunately, the potential for collecting data on large elasmobranchs is increasing. This is due in part to the work of organizations such as OCEARCH [125] that provide expertise and resources for sampling large animals safely and humanely. Such work can coordinate diverse sampling goals, allowing microbiome data to be coupled to host and environmental parameters. Elasmobranchs caught in fisheries can also be sampled for microbiome analysis. However, the potential for microbiomes to change rapidly after death could bias data from fisheries-captured elasmobranchs. Access to live specimens is therefore vital, as is ensuring that organisms are captured and released safely and humanely. Ideally, microbiome sampling of live animals should be paired with sampling of host physiology (e.g., fatty acid profiles, heavy metal concentrations, oxygen consumption, or reproduction status) to establish the role of the microbiome in host health.

Kee** individuals under managed care creates opportunities for experimentation and microbiome sampling over time. The latter is valuable for assessing microbiome stability and would ideally be coupled with measurements of host physiology and environmental conditions, including characterizations of the seawater microbiome. Holistic datasets of this sort would allow researchers to distinguish residents from transient microbiome members, quantify the degree to which the microbiome is affected by environmental and host factors (eg., diet shifts, disease), and identify those microbial taxa most relevant to host health. Though valuable, studies of individuals under managed care present challenges. Notably, many elasmobranchs, particularly larger species, can be hard to house in aquaria. There also is no guarantee that conclusions drawn from these animals apply to those in the wild. Despite these caveats, academic and commercial aquariums have had long term success in maintaining healthy elasmobranchs. These institutions often maintain detailed animal health and diet records and may engage in conservation and veterinary research that could easily integrate a microbiome component. Standardization of microbiome sampling methods across institutions could be relatively straightforward and would enable comparisons across diverse aquaria-housed species, environmental conditions, and potential changes in host disease state. Collecting microbiome samples from aquaria-housed elasmobranchs is relatively non-invasive and inexpensive and should be considered in monitoring and time-series research plans to understand host health.

Elasmobranch microbiomes have thus far been understood primarily through marker gene surveys targeting the phylogenetically informative 16S rRNA gene. These surveys provide valuable insight into community taxonomic diversity. However, these surveys only infer, but do not confirm, the ecological roles of microbiome members based on the assumption that a microbe’s function is aligned with its phylogenetic placement. However, horizontal gene transfer, genomic scavenging, and phage infection can change the ecological role of a microbial strain [126]. Shotgun sequencing of community DNA (metagenomics) characterizes both taxonomically informative marker genes and protein-coding metabolic genes and thereby provides insight into the ecological potential of a microbiome. While this method is widely used in microbiome research in general (e.g., [127]), it has thus far been applied in a small number of elasmobranch microbiome studies. These studies have revealed microbiome-host co-diversification [106], metabolic functions enriched in elasmobranch microbiomes [9], and a large proportion of microbiome protein-coding sequences without clear homologs in databases [9]. Future work to more precisely identify the phylogenetic and functional diversity of these sequences may benefit from assembling individual genomic units from metagenome datasets (Metagenome-Assembled Genomes (MAGs); [128]). Such studies have the potential to also provide insight into the host’s genomics. For example, shotgun sequencing of community DNA from the skin of the common thresher allowed reconstruction of the host mitochondrial genome, hel** to clarify the position of this species in the elasmobranch phylogeny [129]. Metagenomic analysis can also characterize other microbiome members, potentially including fungi, other small eukaryotic organisms, and viruses. Viruses/phage are of particular interest given their role in other systems as modulators of host cell metabolism [130] and drivers of bacterial diversity [131] through processes such as classical predatory–prey relationships [132], but have yet to be characterized in elasmobranch microbiomes.

Future elasmobranch microbiome studies, focused on both wild individuals and those under managed care, should continue to measure community taxonomic composition (16S rRNA gene analysis) but also apply metagenomics and other steps to identify the ecological importance of microbiomes from different body niches. For the intestinal microbiome, metagenome sequencing coupled with metabolomic and diet analysis could identify microbial enzymes or metabolites with roles in host nutrition and energy provisioning, waste or osmolyte processing (e.g., urea/nitrogen cycling), and signaling to the host immune system. The natural variation in diet and feeding strategy (e.g., feasting and fasting vs. grazing) in elasmobranchs creates opportunities to test how such factors influence (or are influenced by) the gut microbiome. Similar analysis of the skin microbiome, potentially comparing wounded versus non-wounded tissue, could be used to test if commensal microbes contribute to the low incidence of wound infection in elasmobranchs, potentially via the production of antimicrobial compounds. Additionally, emerging techniques such as CLASI-FISH (combinatorial labelling and spectral imaging—fluorescence in situ hybridization) can be used to visualize the spatial organization of microbial taxa in biofilms and therefore help identify microbe–microbe interactions in the mucus layer of elasmobranch skin [133]. Other visualization techniques such as scanning electron microscopy can also provide critical insight into how microbes interact physically with elasmobranchs, such as showing how dermal denticle structure influences the colonization and arrangement of bacteria in the mucus layer. Finally, the potential for a blood microbiome in healthy elasmobranchs remains intriguing, but thus far unconfirmed. Prior to investigating the biochemical importance of a blood microbiome, additional studies are necessary to show unequivocally that microbes detected in or cultured from blood are not contaminants and are present at higher frequencies than in other aquatic vertebrates sampled using the same methods. If this can be shown, follow-up questions should explore how these microorganisms interact with host physiology to avoid a strong immune response.

We hypothesize that the unique physiology and behavior of elasmobranchs supports novel microbe–host interactions. Recently, for example, the biofluorescent properties of swell sharks (Cephaloscyllium ventriosum) and chain catsharks (Scyliorhinus retifer) have been linked to unique brominated tryptophan–kynurenine metabolites, which have antimicrobial properties [134]. Whether and how such adaptations affect (or are affected by) the microbiome remains to be tested. The rapidly advancing pace of elasmobranch microbiome research suggests exciting discoveries in the next decade. Future exploration of these unique microbial ecosystems may identify novel microbial taxa, compounds (e.g., antibiotics), or mechanisms of microbe–immune system crosstalk, as well as inform questions at the interface of elasmobranch–microbe–human interaction (e.g., treatment protocols for shark bite and stingray barb victims, strategies for managed care). Such research has the potential to establish elasmobranchs as important models for animal microbiome science.