Colonization of gut microbiomes during early life can shape metabolism and immunity of adult animals. However, most data are derived from antibiotic‐treated or germ‐free laboratory mammals. Furthermore, few studies have explored how microbial colonization during critical windows influences a suite of other fitness‐related traits in wild animals. This study tested whether hatching constitutes a critical developmental window for gut microbiome colonization in wild‐caught amphibians and whether perturbations to gut microbiota at hatching shape fitness‐related traits of larval growth, metabolism, metamorphosis and disease susceptibility. We sterilized wood frog eggs and then inoculated them with microbes from differing sources, including from another species (bullfrogs) that differ in disease resistance and life history. We measured development, growth and metabolic rates through metamorphosis among individuals from each microbial treatment. A separate group was exposed to an LD50 dose of ranavirus—an emerging disease—to test for microbiome effects on disease susceptibility. We also quantified rates of deformities to test for microbial treatment effects on overall health. Manipulation of microbiota on eggs altered the trajectory of gut microbiome communities across larval ontogeny, though disruption appeared to be transitory. While microbiome structure converged among the treatments by metamorphosis, the effects of disruption on host phenotypes persisted. Larvae inoculated with the bullfrog gut microbiota exhibited accelerated growth and development rates compared to controls. By contrast, sterilized larvae maintained in sterile water for several days after hatching exhibited greater disruption to their gut microbiota across ontogeny, as well as altered metabolism, more tail deformities, and were more likely to die when exposed to an LD50 dose of ranavirus compared to the other treatments. These results suggest perturbations to the microbiota during critical developmental windows can alter the trajectory of the gut microbiome, and have long‐term effects on fitness‐related traits in larval amphibians. These results suggest that explicit tests of how changes in the composition and abundance of the microbial community shape phenotypes across ontogeny in amphibians could shed light on host–microbe interactions in wildlife, as well as inform conservation efforts to mitigate emerging diseases.

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Exploration of the importance of developmental windows for microbial colonization in diverse animal taxa, and tests of how these shape both animal microbiomes as well as host phenotypes promise to shed needed light on host-microbe interactions. The aims of this study were to explore how gut microbiota diversity of larval amphibians varies among species and across ontogeny, and to test if manipulation of gut colonization can reveal how microbiomes develop. We found that gut microbiomes differ among species and change across larval ontogeny, with distinctive differences between larvae, metamorphic animals, and juvenile frogs. Through applying a gnotobiotic protocol to eggs and cross-inoculating gut microbiomes between species, we demonstrated that microbiota can be transplanted among species and developmental stages. These results also demonstrated that microbial colonization at hatching is potentially formative for long term composition and function of amphibian gut microbiomes, suggesting that hatching may be a critical developmental window for colonization, similar to the effects of birth mode on human microbiomes. Specifically, our results suggest that either the egg jelly and/or capsules surrounding amphibian eggs are likely important sources for initial microbiome inoculation. Furthermore, we speculate these results suggest that vertical transmission may be important to amphibian microbiome establishment and development, as is common among many animal taxa. Taken together, our results suggest that explicit tests of how host developmental windows influence microbial colonization, and shape amphibian microbiomes across life stages promise to provide insight into the ecological and evolutionary dynamics of host-microbe interactions.

Variation in environmental conditions during larval life stages can shape development during critical windows and have lasting effects on the adult organism. Changes in larval developmental rates in response to environmental conditions, for example, can trade off with growth to determine body size and condition at metamorphosis, which can affect adult survival and fecundity. However, it is unclear how use of energy and nutrients shape trade-offs across life-stage transitions because no studies have quantified these costs of larval development and metamorphosis. We used an experimental approach to manipulate physiological stress in larval amphibians, along with respirometry and C-breath testing to quantify the energetic and nutritional costs of development and metamorphosis. Central to larval developmental responses to environmental conditions is the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis, which regulates development, as well as energy homeostasis and stress responses across many taxa. Given these pleiotropic effects of HPA/I activity, manipulation of the HPA/I axis may provide insight into costs of metamorphosis. We measured the energetic and nutritional costs across the entire larval period and metamorphosis in a larval amphibian exposed to exogenous glucocorticoid (GC) hormones - the primary hormone secreted by the HPA/I axis. We measured metabolic rates and dry mass across larval ontogeny, and quantified lipid stores and nutrient oxidation viaC-breath testing during metamorphosis, under control and GC-exposed conditions. Changes in dry mass match metamorphic states previously reported in the literature, but dynamics of metabolism were influenced by the transition from aquatic to terrestrial respiration. GC-treated larvae had lower dry mass, decreased fat stores and higher oxygen consumption during stages where controls were conserving energy. GC-treated larvae also oxidized greater amounts of C-labelled protein stores. These results provide evidence for a proximate cause of the physiological trade-off between larval growth and development, and provide insight into the energetic and nutrient costs that shape fitness trade-offs across life stages.

Abstract1. Ubiquitous environmental stressors are often thought to alter animal susceptibility to pathogens and contribute to disease emergence. However, duration of exposure to a stressor is likely critical, because while chronic stress is often immunosuppressive, acute stress can temporarily enhance immune function. Furthermore, host susceptibility to stress and disease often varies with ontogeny; increasing during critical developmental windows. How the duration and timing of exposure to stressors interact to shape critical windows and influence disease processes is not well tested.2. We used ranavirus and larval amphibians as a model system to investigate how physiological stress and pathogenic infection shape development and disease dynamics in vertebrates. Based on a resource allocation model, we designed experiments to test how exposure to stressors may induce resource trade-offs that shape critical windows and disease processes because the neuroendocrine stress axis coordinates developmental remodelling, immune function and energy allocation in larval amphibians.3. We used wood frog larvae (Lithobates sylvaticus) to investigate how chronic and acute exposure to corticosterone, the dominant amphibian glucocorticoid hormone, mediates development and immune function via splenocyte immunohistochemistry analysis in association with ranavirus infection. 4. Corticosterone treatments affected immune function, as both chronic and acute exposure suppressed splenocyte proliferation, although viral replication rate increased only in the chronic corticosterone treatment.5. Time to metamorphosis and survival depended on both corticosterone treatment and infection status. In the control and chronic corticosterone treatments, ranavirus infection decreased survival and delayed metamorphosis, although chronic corticosterone exposure accelerated rate of metamorphosis in uninfected larvae. Acute corticosterone exposure accelerated metamorphosis increased survival in infected larvae.6. Interactions between stress exposure (via glucocorticoid actions) and infection impose resource trade-offs that shape optimal allocation between development and somatic function. As a result, critical disease windows are likely shaped by stress exposure because any conditions that induce changes in differentiation rates will alter the duration and susceptibility of organisms to stressors or disease. K E Y W O R D Samphibian, corticosterone, glucocorticoid, larvae, metamorphosis, ranavirus

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Behavioural phenotypes may provide a means for identifying individuals that disproportionally contribute to disease spread and epizootic outbreaks. For example, bolder phenotypes may experience greater exposure and susceptibility to pathogenic infection because of distinct interactions with conspecifics and their environment. We tested the value of behavioural phenotypes in larval amphibians for predicting ranavirus transmission in experimental trials. We found that behavioural phenotypes characterized by latency-to-food and swimming profiles were predictive of disease susceptibility and infectiousness defined as the capacity of an infected host to transmit an infection by contacts. While viral shedding rates were positively associated with transmission, we also found an inverse relationship between contacts and infections. Together these results suggest intrinsic traits that influence behaviour and the quantity of pathogens shed during conspecific interactions may be an important contributor to ranavirus transmission. These results suggest that behavioural phenotypes provide a means to identify individuals more likely to spread disease and thus give insights into disease outbreaks that threaten wildlife and humans.

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