Ectotherm heat tolerance and the microbiome: current understanding, future directions and potential applications

Metazoans form complex relationships with the communities of microbes (e.g. bacteria, fungi, archaea, viruses) that live in and on their bodies, collectively termed the microbiome (see Glossary). Over evolutionary time, hosts have come to rely on these microbes for proper physiological functioning, including digestive efficiency, immune function, metabolic signaling and animal development (Kohl and Carey, 2016; McFall-Ngai et al., 2013). The effects of the microbiome on host physiology can further affect how animals interact with their external environments. Well-known examples include the breakdown of plant material and provisioning of nutrients facilitating herbivorous diets (Dearing and Kohl, 2017; Mandal et al., 2015), and competition with invading pathogens to prevent infection (Kamada et al., 2013). More recently, it is becoming clear that relationships with host-associated microbes influence the response of ectothermic hosts to their thermal environment, specifically by increasing their tolerance to heat (Hector et al., 2022). The implications of these findings are especially important because global climate change presents one of the biggest conservation challenges of our time, and ectotherms are expected to be highly vulnerable to the deleterious effects of increasing temperatures (Jørgensen et al., 2022). In this Commentary, we aim to create a unified view of this growing field of research that will inform efforts to understand how microbes shape the ecological and evolutionary responses of ectotherms to their thermal environments. We provide a brief background of what is known regarding the impact of the microbiome on ectotherm heat tolerance, summarize areas of future research to be prioritized, and consider the possibility of manipulating microbes to achieve desired conservation outcomes.

The relationship between host-associated microbes and heat tolerance is currently best understood in invertebrate hosts. For example, in marine invertebrates, colonization of coral with heat-tolerant bacteria allows hosts to persist in otherwise thermally extreme habitats (Ziegler et al., 2017), and the use of probiotics may be a promising tool to help mitigate the devastating effects of coral bleaching (Rosado et al., 2019). Transfer of microbes from heat-acclimated sea anemones to non-acclimated individuals increases the survival of the recipients under heat stress (Baldassarre et al., 2022). In insects, bacterial endosymbionts (see Glossary) have commonly been shown to elevate their host's tolerance to heat. For example, in the aphid Acyrthosiphon pisum, colonization with certain secondary endosymbionts can increase host growth rate, survival and fecundity under heat stress (Harmon et al., 2009; Montllor et al., 2002; Russell and Moran, 2006). Similarly, in the whitefly Bemisia tabaci, colonization with the secondary endosymbiotic bacteria Rickettsia reduces heat-induced mortality rates of the host (Brumin et al., 2011). In addition to bacterial symbionts, colonization with viruses can also increase insect host heat tolerance. For example, colonization with their host plant's viral pathogen results in a dramatic increase in the critical thermal maximum (CT_max, see Glossary) of the aphid Rhopalosiphum padi (Porras et al., 2020), and increased survival under heat stress in the whitebacked plant-hopper, Sogatella furcifera (Xu et al., 2016). These viruses may even influence the behavioral thermal preference of insects, as colonized aphids prefer warmer regions of host plants than uncolonized individuals (Porras et al., 2020). Whole communities of host-associated microbes, rather than single symbionts, may also influence the heat tolerance of insect hosts. In Drosophila fruit flies, transfer of bacterial communities from the gut of cold-adapted flies decreases CT_max of the recipients (Moghadam et al., 2018) and axenic (see Glossary) individuals show reduced survival under heat stress (Jaramillo and Castañeda, 2021). In an invertebrate roundworm, Caenorhabditis elegans, colonization of the worm through feeding with certain bacterial strains can enhance host fecundity (Hoang et al., 2019) and survival (Nakagawa et al., 2016) under heat stress, and facilitate adaptation of the host to hotter environments (Hoang et al., 2021). The mechanisms responsible for the impact of the microbiota on invertebrate host heat tolerance have been shown to include: (1) the microbial stimulation of host expression of genes with a role in stress response pathways, including those involving heat shock proteins, antioxidant resistance and the cytoskeleton (Brumin et al., 2011; Nakagawa et al., 2016; Porras et al., 2020), and (2) the direct action of microbially produced metabolites and proteins, including microbial heat shock proteins (Dunbar et al., 2007), as a well as a diversity of metabolites that may be released to the host upon heat-induced lysis of microbial cells (Burke et al., 2010).

Relationships between the microbiome and host heat tolerance have been less well studied in ectothermic vertebrate hosts. In lizards, some studies have correlated aspects of the gut microbiome with host heat tolerance. Specifically, a principal coordinate axis representing composition of the bacterial gut microbiota is associated with the CT_max of western fence lizards (Moeller et al., 2020). In common lizards, experimental warming reduces the diversity of the bacterial gut microbiome and this loss of microbial diversity is subsequently associated with reduced animal survival (Bestion et al., 2017). In tadpoles, a direct link has been established between host-associated microbes and host heat tolerance. To experimentally disrupt the microbiome, tadpoles were housed in sterilized water, which significantly reduces the diversity and changes the composition of the bacterial gut microbiome (Fontaine et al., 2022). This treatment is also likely to result in disruption to microbes on other body surfaces, such as the skin (Knutie et al., 2017). Tadpoles with a disrupted microbiome show reductions in host CT_max, loss of locomotor performance at high temperatures and poorer survival under heat stress compared with tadpoles colonized with more natural microbiota (Fontaine et al., 2022). Although the mechanisms responsible for these relationships in vertebrates are far less resolved than those in invertebrates, they may involve oxygen limitation in accordance with the oxygen- and capacity-limited thermal tolerance hypothesis (see Glossary) (Pörtner, 2001). Specifically, tadpoles with a disrupted microbiome exhibit lower activities of oxidative mitochondrial enzymes and increased resting metabolic rate (see Glossary) under warming conditions compared with those with more natural microbiota, suggesting they could be more oxygen limited (Fontaine et al., 2022). A secondary hypothesis suggests that amino acid metabolism could underlie the impact of the microbiome on host heat tolerance. In a subsequent experiment with tadpoles, under warm conditions, when compared with tadpoles with disrupted microbiota, hosts with a more natural microbiota upregulated more genes related to amino acid biosynthesis and catabolism, and had microbiomes that were enriched for amino acid production (Fontaine and Kohl, 2023). Previous studies in other ectothermic systems have shown an accumulation of free amino acids – specifically branched chain amino acids – during heat stress, suggesting that proteins are an important energy source used by organisms to maintain metabolism during warming (Tripp-Valdez et al., 2017). This process may explain the association between microbially mediated changes in amino acid metabolism and host heat tolerance.

In Box 1, we highlight several important and unanswered questions relating to the interaction between the microbiome and the heat tolerance of ectothermic hosts. Each of the relevant areas are discussed in further detail below.

One important path forward in this field involves broadening study systems to a wider variety of hosts. There have been studies across many invertebrate host species that aim to understand the impacts of their associated microbes on host heat tolerance and performance, and the mechanisms behind these effects; however, much work is still needed to understand these relationships in ectothermic vertebrate hosts (Hector et al., 2022). To our knowledge, effects of the microbiome on host responses to heat in ectothermic vertebrates have been studied in two lizard species (Bestion et al., 2017; Moeller et al., 2020) and a single species of tadpole (Fontaine and Kohl, 2023; Fontaine et al., 2022). Studies in more ectothermic vertebrate hosts, including a wider variety of reptile and amphibian species, and fish, will clarify how commonly the microbiome has an effect on thermal biology in this group of animals. Additionally, to our knowledge, no studies have focused on whether, and how, the relationship between microbes and heat tolerance changes temporally throughout an animal's life cycle. Specifically, in animals with complex life cycles, such as amphibians or insects, it will be important to understand whether influences of the larval microbial environment on host heat tolerance persist in the adult stage, as has been shown for microbially mediated parasite susceptibility (Knutie et al., 2017). Further, the mechanisms delimiting thermal tolerance can differ between aquatic and terrestrial habitats (Verberk et al., 2016); thus, studies in both habitat types may help to uncover whether the microbiome impacts thermal tolerance similarly in these different contexts, as we are only currently aware of a single study in an aquatic system (Fontaine et al., 2022). Likewise, dietary strategy (i.e. herbivorous versus carnivorous) affects the thermal sensitivity of ectotherms (Hardison et al., 2023), as well as microbiome composition (Leigh et al., 2022): an additional area of research could be to understand how relationships between microbes and thermal tolerance differ across dietary habits, as most studies have been conducted in herbivorous animals. Not all animals house diverse communities of microbes, and microbes may not always be important to host physiology (Hammer et al., 2019); thus, broadening our efforts to more diverse study systems will help us understand when microbes do and, importantly, do not impact host heat tolerance.

In addition to host systems, the field may also benefit from broadening the microbial diversity studied in these associations. Across all hosts, most studies focus on bacteria (except for a few viral studies, see Introduction) living in the gut or as intracellular endosymbionts (see Introduction). Other groups of microbes, such as fungi, and those living in other organs, such as the skin, play crucial roles in host physiology (Chin et al., 2020; Grice, 2015); however, we currently lack understanding of how they may contribute to host heat tolerance.

Further, more mechanistic studies are needed across a variety of hosts to understand the drivers of the relationship between microbial communities and host thermal performance in different contexts. Particularly, we lack mechanistic studies in vertebrate hosts. Although mechanisms could be similar between invertebrates and vertebrates, most mechanistic studies in invertebrate hosts have been performed in systems with insect hosts and endosymbiotic bacteria (see Introduction). Endosymbionts are transmitted vertically, provision specific nutrients to their hosts and maintain a long history of co-evolution with their hosts (Engel and Moran, 2013). Thus, the reliance of the host on symbionts in these cases, and the mechanisms by which microbes can influence their host's physiology may differ significantly from microbes in vertebrate-associated communities which are commonly transmitted horizontally and experience high degrees of change (Priya and Blekhman, 2019). Focus could be directed towards understanding how the microbiome affects host thermal tolerance in vertebrate systems, as well as in invertebrate systems where more complex communities have been associated with host heat tolerance, such as flies (Jaramillo and Castañeda, 2021; Moghadam et al., 2018). Thus far in vertebrates, in correlative studies, whole-compositional aspects of the microbiome, such as microbiome beta diversity (see Glossary; Moeller et al., 2020) and alpha diversity (see Glossary; Bestion et al., 2017) have been associated with vertebrate host responses to heat; however, in manipulative studies, water sterilization techniques have been used to manipulate the microbiome, which alters microbiome diversity, overall microbiome composition, and the relative abundance of many bacterial taxa simultaneously (Fontaine et al., 2022). Thus, it is currently unknown which of these changes in the microbiome is truly responsible for the impacts on host heat tolerance. Disentangling these effects is necessary to understand the mechanism behind these relationships. Future studies may focus on more targeted microbial manipulations, such as varying microbial community composition and diversity independently of one another, as well as supplementing or removing specific microbial taxa in the community, to isolate the microbial phenotype responsible for effects on host thermal performance.

Studies manipulating an animal's external environmental conditions may also help to elucidate the mechanisms driving these relationships. As described above, there is evidence that the link between the tadpole microbiome and host heat tolerance could be driven by oxygen limitations or changes in amino acid availability in animals with disrupted microbiomes (Fontaine and Kohl, 2023; Fontaine et al., 2022). Thus, manipulating levels of available oxygen or dietary inputs of amino acids, including both the quantity and identity of specific amino acids, and observing the changes in thermal tolerance in individuals with varying microbiome compositions could shed light on which mechanisms are responsible.

In addition to mechanistic studies, future studies may focus on replicating more natural conditions to understand whether host-associated microbial communities actually affect host thermal tolerance in the wild. The majority of studies (invertebrate and vertebrate) that have discovered relationships between animal-associated microbes and host responses to heat have done so in the laboratory (Fig. 1; Brumin et al., 2011; Fontaine et al., 2022; Harmon et al., 2009; Hoang et al., 2019, 2021; Jaramillo and Castañeda, 2021; Moeller et al., 2020; Moghadam et al., 2018; Montllor et al., 2002; Nakagawa et al., 2016; Porras et al., 2020; Russell and Moran, 2006), although in more natural mesocosm enclosures, loss of microbiota diversity due to warming was shown to be associated with reduced survival in lizards (Bestion et al., 2017). Natural environments are far more complex than laboratory environments, and captivity can dramatically alter the diversity and composition of host-associated microbial communities compared with wild microbiomes (Kohl et al., 2017; McKenzie et al., 2017). Thus, it is difficult to know from laboratory studies whether the impacts of the microbiome on host heat tolerance would be consistent in animals exposed to much more biotic and abiotic environmental variation and when colonized with more wild-type communities (Fig. 1). Furthermore, in the wild, there may be tradeoffs between the positive effects of microbiome-induced increases in heat tolerance with other phenotypes caused by alterations to microbial communities. For example, in tadpoles, disrupting the microbiome causes animals to be less heat tolerant, but also substantially increases animal body size (Fontaine et al., 2022), which could have positive impacts on animal fitness in nature (Cabrera-Guzmán et al., 2013). In aphids, colonization with particular heat-resistant symbionts increases host growth rate under heat shock conditions, but reduces host growth rate under non-stressful conditions (Harmon et al., 2009), indicating that the association may only confer benefits in certain contexts. Wild studies that capture the full range of environmental conditions faced by hosts will help to elucidate when the benefits of colonization with heat-protective microbes outweigh the costs (Fig. 1). This goal could be accomplished through realistic mesocosm experiments, such as those performed by Bestion et al. (2017), in which the impact of climate warming on lizard gut microbiota was assessed through maintaining animals in seminatural outdoor enclosures in which temperature could be manipulated, but individuals still had opportunities to perform natural behaviors, such as thermoregulation and foraging. Additionally, experiments could utilize targeted microbial treatments of animals followed by release into the wild and subsequent tracking of fitness, following similar methods to studies that have assessed the efficacy of disease treatments in natural populations (Hudson et al., 2016). These types of experiments will be most relevant to understanding how interactions with microbes will affect susceptibility to climate change in wild populations.

Another intriguing direction could be to more accurately disentangle the distinct or interacting contributions of the host versus the microbiome to thermal tolerance. This is also in alignment with a push to expand our understanding of the full determinants of organismal phenotypes (Koskella and Bergelson, 2020). In the past, phenotypes have been reasoned to be influenced by genotype (G), environment (E) and genotype×environment interactions (G×E). However, there is now reason to incorporate the microbiome (M) to understand the potential for complex interactions among these factors (G×E×M) (Oyserman et al., 2021). The thermal tolerance of many ectothermic species varies geographically (Guiterrez-Pesquera et al., 2016; Sunday et al., 2011), and can also covary with membership of the gut microbiome (Moeller et al., 2020). Further, a number of ectothermic species, such as the stinkbug Riptortus clavatus (Kikuchi et al., 2007) and the bobtail squid Euprymna scolopes (McFall-Ngai, 2014), acquire microbial symbionts from their environments, and in several fish species (Wu et al., 2012, 2013; Wong et al., 2015), the gut microbiota largely reflects those microbes present in their surrounding environments. Work in microbial ecology has also shown global patterns in some environmental microbiomes, such as covariation of global soil microbial diversity with temperature (Gillingham et al., 2019). Thus, there is potential for variation in the interactions between hosts and environmentally acquired microbes to influence host phenotypes (Bo and Kohl, 2021). For example, in the aphid A. pisum, colonization with the secondary bacterial endosymbiont Serratia symbiotica increases host fecundity and development under heat shock, but S. symbiotica strains from warm Arizona increase these phenotypes to a greater degree than strains from cooler New York or Wisconsin (Russell and Moran, 2006).

Given the goal of predicting species' responses to climate change, there is interest in understanding the different contributions of local host adaptation, local microbial adaptation and the potential need for microbes and hosts to work collaboratively to overcome thermal stress (Fig. 1; Fontaine and Kohl, 2020b, 2023). Work in this area will also expand our understanding of how natural selection may act on the holobiont (see Glossary) (Theis et al., 2016; Zilber-Rosenberg and Rosenberg, 2008). These interactions have been best demonstrated thus far in a sea anemone (Nematostella vectensis) system; genotypes for this species were collected across a North–South gradient, and anemones were grown under various temperatures in common laboratory environments. Researchers observed significant effects of genotype (G) and temperature (E) on microbiome composition, but also saw a significant G×E interaction in shaping the microbiome of these organisms (Baldassarre et al., 2023). The microbiome of these animals also offers a source of rapid adaptation, as the transfer of microbes from heat-acclimated N. vectensis can increase thermal tolerance in recipients (Baldassarre et al., 2022). These results demonstrate the potential for the host, microbiome and their interactions to determine the evolutionary potential of collective holobionts (Henry et al., 2021).

Climate change is one of the most pressing issues facing wildlife worldwide. Ectotherms in particular are expected to experience disproportionate increases in mortality due to rising mean and maximum environmental temperatures (Jørgensen et al., 2022). Climate change can also result in sublethal effects on ectotherm physiology (Dillon et al., 2010), behavior (Kearney et al., 2009) and reproduction (Bestion et al., 2015). Recently, it has become clear that increasing temperatures can additionally affect ectotherms through changes in their associated microbial communities. Exposure to high temperatures changes the community composition of the microbiome and/or reduces its diversity in ectotherms such as corals (Ziegler et al., 2017), insects (Arango et al., 2021; Moghadam et al., 2018), fish (Kokou et al., 2018), amphibians (Fontaine and Kohl, 2020a; Fontaine et al., 2022, 2018; Kohl and Yahn, 2016; Zhu et al., 2021) and reptiles (Bestion et al., 2017; Moeller et al., 2020); however, in some cases, warming may increase microbial diversity (Kokou et al., 2018) or have no effect on the microbiome at all (Williams et al., 2022). The effect of heat on the microbiome sets up the possibility for positive-feedback loops in which environmental warming may decrease ectotherm heat tolerance and increase their vulnerability to warming (Fig. 2). For example, raising tadpoles in sterilized water alters the community composition and reduces diversity of the bacterial gut microbiome compared with that of tadpoles with more natural microbiota; subsequently, this reduces their heat tolerance (Fontaine et al., 2022). However, increasing the tadpoles' rearing temperature by 6°C decreases microbial diversity in the natural microbiota group to the same degree as in the tadpoles reared in sterilized water (Fontaine et al., 2022). If a loss of microbial diversity is the main driver of reduced heat tolerance, these results suggest that warming-induced losses of diversity could further lower host thermal tolerance, leaving them more vulnerable to climate warming; this may compound over time. However, hosts also acclimate to warm temperatures, which typically increases host heat tolerance (Brattstrom and Lawrence, 1962); thus, the degree to which warming-induced loss of microbial diversity and warming-induced host physiological acclimation contribute to changes in overall heat tolerance warrants future study. This goal could be accomplished through comparing the heat tolerance of axenic versus microbially colonized animals following acclimation to warm or control temperatures, or by utilizing microbial transplants among hosts with varying genotypes to isolate specific host versus microbial contributions to thermal acclimation. Similar experiments have been conducted in the sea anemone system described above (Baldassarre et al., 2022) and in endothermic laboratory mice to uncover the role of the microbiome in host thermogenesis (Chevalier et al., 2015; Li et al., 2019).

If specific microbes or microbial compositions can be identified that bolster host heat tolerance, it may be important to direct efforts to monitoring and preserving the diversity of wildlife-associated microbial communities. Although conservation biology has typically focused on host animal and habitat preservation, these microbial communities may be harnessed for conservation applications through bioaugmentation approaches (see Glossary; Trevelline et al., 2019). Probiotic treatments, in which specific ‘beneficial’ microbes are administered as a protective treatment, are commonly used in agriculture, aquaculture and human medicine to achieve desired outcomes; these are becoming increasingly common in wildlife settings, particularly to protect hosts from disease (McKenzie et al., 2018). For example, several bacteria found on amphibian skin are capable of inhibiting the fungal pathogen Batrachochytrium dendrobatidis (Becker et al., 2015), and development of these microbes into probiotics to protect hosts from disease has been an active area of research for some time (Bletz et al., 2013). Already in development in marine systems (Rosado et al., 2019), probiotic techniques could also be applied to protect hosts from climate change. This approach would require identification of specific microbial taxa that are associated with increased heat tolerance in hosts, followed by their cultivation in the laboratory (Fig. 1). If these microbes are from the gastrointestinal tract, successful culture could pose a challenge, but new ‘culturomics’ techniques have emerged allowing for cultivation of a wide variety of gut microbes in the laboratory (Lagier et al., 2016). Individual or mixtures of microbes could then be administered to hosts, followed by verification that the treatment results in increased heat tolerance. If whole-compositional aspects of the microbiome (rather than specific taxa) are associated with heat tolerance (Moeller et al., 2020), whole microbiomes could be transplanted through fecal microbiome transplants (FMTs), in which feces from donor individuals are transplanted into recipient individuals with the goal of transferring important microbes (Borody et al., 2013). This approach is commonly used to prevent disease in human medicine (Antushevich, 2020), and the transfer of microbes between hosts can change thermal tolerance in invertebrate hosts (Baldassarre et al., 2022; Moghadam et al., 2018). Donor individuals could be chosen by identification of hosts with the highest heat tolerance or by selection of hosts from warmer habitats where symbionts locally adapted to heat are likely to occur (Russell and Moran, 2006).

Regardless of whether probiotics or FMTs are used, there are several considerations that would need to be addressed before using these approaches as widespread treatments. For example, do the probiotic or transplanted microbes colonize and persist in the gut, or do they transiently pass through? Both host factors and characteristics of the resident microbiome affect probiotic persistence (Maldonado-Gómez et al., 2016), and aspects of the recipient's external environment (e.g. temperature) are likely to affect the maintenance of these microbes in the gut (Bletz et al., 2013). Additionally, the majority of wildlife bioaugmentation studies have been performed in laboratory settings (Bletz et al., 2013); thus, once treatments have shown the ability to bolster host heat tolerance in the lab, well-designed field trials will be needed to demonstrate their ability to maintain this affect without adverse consequences in nature (Fig. 1). Nevertheless, once these hurdles are cleared, bioaugmentation strategies could represent an important tool to protect hosts that are particularly vulnerable to climate warming because of their physiology (Somero, 2010) or the habitat they occupy (Deutsch et al., 2008).

Although we are rapidly gaining an understanding of the role that microbes play in various physiological processes – including the thermal tolerance of ectotherms – much remains to be uncovered. Addressing the questions outlined here will enhance our understanding of how natural selection interacts with the holobiont, and how physiological tolerance is determined by hosts, microbiomes and/or distinct interactions within these partnerships. Additionally, work in this area will shed light on the speed at which mutualisms might facilitate rapid ecological and evolutionary adaptations. Although it currently seems to belong to a distant future, once we have this understanding in hand, we may be able to act as better stewards of the Earth's resources through conservation actions that involve the use of probiotics and other microbial manipulations to enhance the thermal tolerance of imperiled ectothermic species. We hope that pursuing the research questions outlined in this Commentary will go some way toward achieving this goal.

We thank the editors and two anonymous reviewers for thoughtful comments that improved this paper.