Cross-species gut microbiota transplantation predictably affects host heat tolerance.

The gut microbiome is known to influence and have regulatory effects in diverse physiological functions of host animals, but only recently has the relationship between host thermal biology and gut microbiota been explored. Here, we examined how early-life manipulations of the gut microbiota in larval amphibians influenced their critical thermal maximum (CTmax) at different acclimation temperatures. We stripped the resident microbiome from egg masses of wild-caught wood frogs (Lithobates sylvaticus) via an antibiotic wash, and then inoculated the eggs with pond water (control), no inoculation, or the intestinal microbiota of another species that has a wider thermal tolerance - green frogs (Lithobates clamitans). We predicted that this cross-species transplant would increase the CTmax of the recipient wood frog larvae relative to the other treatments. In line with this prediction, green frog microbiome-recipient larvae had the highest CTmax while those with no inoculum had the lowest CTmax. Both the microbiome treatment and acclimation temperature significantly influenced the larval gut microbiota communities and α-diversity indices. Green frog microbiome-inoculated larvae were enriched in Rikenellaceae relative to the other treatments, which produce short-chain fatty acids and could contribute to greater energy availability and enhanced heat tolerance. Larvae that received no inoculation had a higher relative abundance of potentially pathogenic Aeromonas spp., which negatively affects host health and performance. Our results are the first to show that cross-species gut microbiota transplants alter heat tolerance in a predictable manner. This finding has repercussions for the conservation of species that are threatened by climate change and demonstrates a need to further explore the mechanisms by which the gut microbiota modulate host thermal tolerance.

Ranavirus infection does not reduce heat tolerance in a larval amphibian.

Extreme heat events and emerging infectious diseases negatively impact wildlife populations, but the interacting effects of infection and host heat tolerance remain understudied. The few studies covering this subject have demonstrated that pathogens lower the heat tolerance of their hosts, which places infected hosts at a greater risk experiencing lethal heat stress. Here, we studied how ranavirus infection influenced heat tolerance in larval wood frogs (Lithobates sylvaticus). In line with similar studies, we predicted the elevated costs of ranavirus infection would lower heat tolerance, measured as critical thermal maximum (CTmax), compared to uninfected controls. Ranavirus infection did not reduce CTmax and there was a positive relationship between CTmax and viral loads. Our results demonstrate that ranavirus-infected wood frog larvae had no loss in heat tolerance compared to uninfected larvae, even at viral loads associated with high mortality rates, which contradicts the common pattern for other pathogenic infections in ectotherms. Larval anurans may prioritize maintenance of their CTmax when infected with ranavirus to promote selection of warmer temperatures during behavioral fever that can improve pathogen clearance. Our study represents the first to examine the effect of ranavirus infection on host heat tolerance, and because no decline in CTmax was observed, this suggests that infected hosts would not be under greater risk of heat stress.

Heat hardening of a larval amphibian is dependent on acclimation period and temperature.

Plasticity in heat tolerance provides ectotherms the ability to reduce overheating risk during thermal extremes. However, the tolerance-plasticity trade-off hypothesis states that individuals acclimated to warmer environments have a reduced plastic response, including hardening, limiting their ability to further adjust their thermal tolerance. Heat hardening describes the short-term increase in heat tolerance following a heat shock that remains understudied in larval amphibians. We sought to examine the potential trade-off between basal heat tolerance and hardening plasticity of a larval amphibian, Lithobates sylvaticus, in response to differing acclimation temperatures and periods. Lab-reared larvae were exposed to one of two acclimation temperatures (15°C and 25°C) for either 3 or 7 days, at which time heat tolerance was measured as critical thermal maximum (CTmax ). A hardening treatment (sub-critical temperature exposure) was applied 2 h before the CTmax assay for comparison to control groups. We found that heat-hardening effects were most pronounced in 15°C acclimated larvae, particularly after 7 days of acclimation. By contrast, larvae acclimated to 25°C exhibited only minor hardening responses, while basal heat tolerance was significantly increased as shown by elevated CTmax temperatures. These results are in line with the tolerance-plasticity trade-off hypothesis. Specifically, while exposure to elevated temperatures induces acclimation in basal heat tolerance, shifts towards upper thermal tolerance limits constrain the capacity for ectotherms to further respond to acute thermal stress.

Captivity and Animal Microbiomes: Potential Roles of Microbiota for Influencing Animal Conservation.

During the ongoing biodiversity crisis, captive conservation and breeding programs offer a refuge for species to persist and provide source populations for reintroduction efforts. Unfortunately, captive animals are at a higher disease risk and reintroduction efforts remain largely unsuccessful. One potential factor in these outcomes is the host microbiota which includes a large diversity and abundance of bacteria, fungi, and viruses that play an essential role in host physiology. Relative to wild populations, the generalized pattern of gut and skin microbiomes in captivity are reduced alpha diversity and they exhibit a significant shift in community composition and/or structure which often correlates with various physiological maladies. Many conditions of captivity (antibiotic exposure, altered diet composition, homogenous environment, increased stress, and altered intraspecific interactions) likely lead to changes in the host-associated microbiome. To minimize the problems arising from captivity, efforts can be taken to manipulate microbial diversity and composition to be comparable with wild populations through methods such as increasing dietary diversity, exposure to natural environmental reservoirs, or probiotics. For individuals destined for reintroduction, these strategies can prime the microbiota to buffer against novel pathogens and changes in diet and improve reintroduction success. The microbiome is a critical component of animal physiology and its role in species conservation should be expanded and included in the repertoire of future management practices.

Microbiome mediation of animal life histories via metabolites and insulin-like signalling.

The regulatory pathways by which gut microbiota potentially shape host life histories remain largely untested, however, a constellation of research suggests that gut bacteria likely have significant effects on their hosts via metabolites. In this article we review known and hypothesized pathways by which gut microbiota influence host life histories through interfacing with the neuroendocrine system, with a focus on the insulin-like growth factor (IGF) signalling pathway. Bacterially derived metabolites including short-chain fatty acids (SCFAs), polyamines, and peptides likely impact host life histories as metabolic substrates, essential nutrients, and via molecular signalling with well-studied neuroendocrine pathways. The hypothalamus-pituitary axis and insulin-like signalling (ILS) pathways are central regulatory networks for development, growth, reproductive maturity, reproduction, and senescence and are likely targets for tests of how gut bacterial metabolites shape host life histories. SCFAs in particular, as metabolites derived from bacterial fermentation, are implicated as significant microbiome signalling molecules shown to interface with the ILS pathway as well as to bind to receptors on neuroendocrine and peripheral nervous tissues. For example, experimental increases of SCFA production have been shown to affect IGF-1 levels in circulation and are associated with robust development, growth, reproduction, and delayed senescence. Finally, emerging -omics approaches are providing integrative ways to test and detail the potential diverse ways in which gut microbiota interact with their hosts and the likely important roles they play in shaping host life-history responses to varied environmental conditions.

Heterothermy as a mechanism to offset energetic costs of environmental and homeostatic perturbations.

Environmental and biotic pressures impose homeostatic costs on all organisms. The energetic costs of maintaining high body temperatures (Tb) render endotherms sensitive to pressures that increase foraging costs. In response, some mammals become more heterothermic to conserve energy. We measured Tb in banner-tailed kangaroo rats (Dipodomys spectabilis) to test and disentangle the effects of air temperature and moonlight (a proxy for predation risk) on thermoregulatory homeostasis. We further perturbed homeostasis in some animals with chronic corticosterone (CORT) via silastic implants. Heterothermy increased across summer, consistent with the predicted effect of lunar illumination (and predation), and in the direction opposite to the predicted effect of environmental temperatures. The effect of lunar illumination was also evident within nights as animals maintained low Tb when the moon was above the horizon. The pattern was accentuated in CORT-treated animals, suggesting they adopted an even further heightened risk-avoidance strategy that might impose reduced foraging and energy intake. Still, CORT-treatment did not affect body condition over the entire study, indicating kangaroo rats offset decreases in energy intake through energy savings associated with heterothermy. Environmental conditions receive the most attention in studies of thermoregulatory homeostasis, but we demonstrated here that biotic factors can be more important and should be considered in future studies.

Taxonomy, not locality, influences the cloacal microbiota of two nearctic colubrids: a preliminary analysis.

BACKGROUND: The gut microbiota is an emerging frontier in wildlife research and its importance to vertebrate health and physiology is becoming ever more apparent. Reptiles, in particular snakes, have not received the same attention given to other vertebrates and the composition of their wild gut microbiome remains understudied. The primary goal of this work was to describe the cloacal microbiota of two Colubrids, the Eastern Gartersnake (Thamnophis sirtalis sirtalis) and the Northern Watersnake (Nerodia sipedon sipedon), and if their cloacal microbiota differed as well as if it did between a wetland and upland population of the former species. METHODS AND RESULTS: We utilized next-generation sequencing of cloacal swabs-a non-destructive proxy for the gut microbiota. The cloacal microbiome of Eastern Gartersnakes (N = 9) was like those of other snakes being comprised of Proteobacteria, Bacteroidetes, and Firmicutes, while that of Northern Watersnakes (N = 6) was dominated by Tenericutes. Seven microbial operational taxonomic units (OTUs), all members of Proteobacteria, were shared among all individuals and were indicative of a core microbiome in Eastern Gartersnakes, but these OTUs were not particularly relevant to Northern Watersnakes. The latter had greater OTU richness than did Eastern Gartersnakes, and habitat did not have any apparent effect on the microbial community composition in Eastern Gartersnakes. CONCLUSIONS: Our findings suggest host taxonomy to be a determining factor in the cloacal microbiota of snakes and that Tenericutes are associated with aquatic habitats. This is the first report to examine the cloacal microbiome of these species and provides a useful foundation for future work to build upon.

Nitrogen stable isotope turnover and discrimination in lizards.

RATIONALE: Nitrogen stable isotope ratio (δ15 N) processes are not well described in reptiles, which limits reliable inference of trophic and nutrient dynamics. In this study we detailed δ15 N turnover and discrimination (Δ15 N) in diverse tissues of two lizard species, and compared these results with previously published carbon data (δ13 C) to inform estimates of reptilian foraging ecology and nutrient physiology. METHODS: We quantified 15 N incorporation and discrimination dynamics over 360 days in blood fractions, skin, muscle, and liver of Sceloporus undulatus and Crotaphytus collaris that differed in body mass. Tissue samples were analyzed on a continuous flow isotope ratio mass spectrometer. RESULTS: Δ15 N for plasma and red blood cells (RBCs) ranged between +2.7 and +3.5‰; however, skin, muscle, and liver did not equilibrate, hindering estimates for these somatic tissues. 15 N turnover in plasma and RBCs ranged from 20.7 ± 4 to 303 ± 166 days among both species. Comparison with previously published δ13 C results for these same samples showed that 15 N and 13 C incorporation patterns were uncoupled, especially during winter when hibernation physiology could have played a role. CONCLUSIONS: Our results provide estimates of 15 N turnover rates and discrimination values that are essential to using and interpreting isotopes in studies of diet reconstruction, nutrient allocation, and trophic characterization in reptiles. These results also suggest that somatic tissues can be unreliable, while life history shifts in nutrient routing and metabolism potentially cause 15 N and 13 C dynamics to be decoupled.

Eurythermic Sprint and Immune Thermal Performance and Ecology of an Exotic Lizard at Its Northern Invasion Front.

AbstractThermal performance of immunity has been relatively understudied in ectotherms, especially in the context of invasive species or in relation to other fitness-related traits and thermoregulatory patterns in the field. For reptiles, thermal biology is a primary factor determining physiological performance and population viability, and suboptimal thermal conditions may limit the expansion of exotic species along the edges of their invasion fronts. This study examined thermoregulatory ecology and thermal performance of immunity and sprinting in a population of Mediterranean geckos (Hemidactylus turcicus) at the northern edge of their invasion front in a temperate zone of the United States. In the field, we quantified temperatures of geckos of varied age classes in relation to air, wall, and refugia temperatures. We also quantified temperature-dependent sprint performance and immune function in field-collected geckos to detail thermal performance patterns that may contribute to the capacity for this species to invade cool climates. Although body temperature (Tb) of wild-caught geckos correlated with wall temperature, average Tb exhibited wide distributions, suggesting eurythermy. Furthermore, the thermal performance of immune swelling responses to phytohemagglutinin injections and sprinting was optimized over a similarly wide temperature range that overlapped with the field Tb's that suggest eurythermy in this species. The wide thermal performance breadths in these traits could buffer against variation in factors such as pathogen exposure and environmental temperatures that could otherwise suppress functional performance. Thus, eurythermy of sprint and immune performance may facilitate the invasive potential of H. turcicus.

Sex and life history shape the strength of cellular and humoral immune responses in a wing dimorphic cricket.

Immune function is a complex collection of responses that often trade-off with one another and with other life history traits, because of the high costs of mounting and maintaining immune responses. Animals, even those from the same populations, may emphasize different aspects of immune function depending on their habitat and phenotype. For example, host population density mediates the threat from density-dependent parasites. Animals at high densities may emphasize fast-acting humoral responses, while those at low densities may favor slower, but more specific, cellular responses. However, these predictions may be dependent on other life history traits, like sex, which is associated with variation in many immune responses. We used wing dimorphic Gryllus firmus crickets to test humoral responses, measured by lysozyme and phenoloxidase activities, and cellular immune responses, measured by encapsulation, between morphs and sex. We found that both morphs and sexes differed in aspects of immune function. Long wing morphs had stronger encapsulation responses than short winged morphs. Additionally, females exhibited higher PO activity than males, and by contrast, males had higher lysozyme activity than females. Our study suggests that G. firmus morphs prioritize different immune responses that may reflect a balancing between the costs of immunity and differing pathogen threats. Male and female crickets exhibit differences in humoral immune responses that may reflect their different life history demands.

Community Physiological Ecology.

The effects of animal homeostatic function on ecological interactions have not been well-integrated into community ecology. Animals mediate environmental change and stressors through homeostatic shifts in physiology and behavior, which likely shape ecological interactions and plant communities. Animal responses to stressors can alter their habitat use, selective foraging, and stoichiometry, which can in turn affect trophic interactions, plant growth, reproduction, and dispersal. Here, we describe a community physiological ecology framework that integrates classical ecological theory and emerging empirical approaches to test how animal homeostatic responses to environmental change mediate ecological interactions and shape communities. Interdisciplinary approaches could provide essential data to characterize and forecast community responses to rapid global environmental change.

Manipulation of gut microbiota during critical developmental windows affects host physiological performance and disease susceptibility across ontogeny.

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.

Critical disease windows shaped by stress exposure alter allocation trade-offs between development and immunity.

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. 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. 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. 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. 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. 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.

Manipulation of Gut Microbiota Reveals Shifting Community Structure Shaped by Host Developmental Windows in Amphibian Larvae.

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.

Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis.

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 13C-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 via 13C-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 13C-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.

Immune function trade-offs in response to parasite threats.

Immune function is often involved in physiological trade-offs because of the energetic costs of maintaining constitutive immunity and mounting responses to infection. However, immune function is a collection of discrete immunity factors and animals should allocate towards factors that combat the parasite threat with the highest fitness cost. For example, animals on dispersal fronts of expanding population may be released from density-dependent diseases. The costs of immunity, however, and life history trade-offs in general, are often context dependent. Trade-offs are often most apparent under conditions of unusually limited resources or when animals are particularly stressed, because the stress response can shift priorities. In this study we tested how humoral and cellular immune factors vary between phenotypes of a wing dimorphic cricket and how physiological stress influences these immune factors. We measured constitutive lysozyme activity, a humoral immune factor, and encapsulation response, a cellular immune factor. We also stressed the crickets with a sham predator in a full factorial design. We found that immune strategy could be explained by the selective pressures encountered by each morph and that stress decreased encapsulation, but not lysozyme activity. These results suggest a possible trade-off between humoral and cellular immunity. Given limited resources and the expense of immune factors, parasite pressures could play a key factor in maintaining insect polyphenism via disruptive selection.

Behavioural phenotypes predict disease susceptibility and infectiousness.

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.

Influence of Physiological Stress on Nutrient Stoichiometry in Larval Amphibians.

Exposure to environmental stressors alters animal phenotypes as well as nutrient metabolism, assimilation, and excretion. While stress-induced shifts in nutrient processes are known to alter organismal carbon (C) and nitrogen (N) stoichiometry, there has been little exploration of how environmental factors influence phosphorous (P). A better understanding of how P cycling varies with animal physiological state may provide insight into across-scale processes, because P is essential to animal function and ecological processes such as production and decomposition. We tested the effects of predator stress and exogenous glucocorticoids on C∶N∶P stoichiometry of larval amphibians. Glucocorticoids altered nutrient stoichiometry, apparently by modulating ossification and renal function. This reduced whole-body P and significantly increased N∶P. Additionally, elevated glucocorticoids caused a long-term reduction in P excretion. This reduction may reflect an initial unmeasured loss of P that glucocorticoids induce over acute timescales. In contrast, exposure to predator cues had no effect on larval C∶N∶P stoichiometry, which highlights that different stressors have varied effects on the endocrine stress response. Predation, in particular, is ubiquitous in the environment; thus, larvae responding to predators have conserved mechanisms that likely prevent or minimize physiological disruption. These results demonstrate the differing physiological roles of N and P, distinct nutrient demands associated with amphibian metamorphosis, and the contrasting effects that different environmental factors have on the physiological stress response. Our results also suggest that anthropogenic changes to the environment that induce chronic stress in amphibians could affect the biogeochemistry of nutrient-poor environments where they may act as keystone species.

Co-Infection by Chytrid Fungus and Ranaviruses in Wild and Harvested Frogs in the Tropical Andes.

While global amphibian declines are associated with the spread of Batrachochytrium dendrobatidis (Bd), undetected concurrent co-infection by other pathogens may be little recognized threats to amphibians. Emerging viruses in the genus Ranavirus (Rv) also cause die-offs of amphibians and other ectotherms, but the extent of their distribution globally, or how co-infections with Bd impact amphibians are poorly understood. We provide the first report of Bd and Rv co-infection in South America, and the first report of Rv infections in the amphibian biodiversity hotspot of the Peruvian Andes, where Bd is associated with extinctions. Using these data, we tested the hypothesis that Bd or Rv parasites facilitate co-infection, as assessed by parasite abundance or infection intensity within individual adult frogs. Co-infection occurred in 30% of stream-dwelling frogs; 65% were infected by Bd and 40% by Rv. Among terrestrial, direct-developing Pristimantis frogs 40% were infected by Bd, 35% by Rv, and 20% co-infected. In Telmatobius frogs harvested for the live-trade 49% were co-infected, 92% were infected by Bd, and 53% by Rv. Median Bd and Rv loads were similar in both wild (Bd = 101.2 Ze, Rv = 102.3 viral copies) and harvested frogs (Bd = 103.1 Ze, Rv = 102.7 viral copies). While neither parasite abundance nor infection intensity were associated with co-infection patterns in adults, these data did not include the most susceptible larval and metamorphic life stages. These findings suggest Rv distribution is global and that co-infection among these parasites may be common. These results raise conservation concerns, but greater testing is necessary to determine if parasite interactions increase amphibian vulnerability to secondary infections across differing life stages, and constitute a previously undetected threat to declining populations. Greater surveillance of parasite interactions may increase our capacity to contain and mitigate the impacts of these and other wildlife diseases.

Pouch brooding marsupial frogs transfer nutrients to developing embryos.

Marsupial frogs have a unique reproductive mode in which females carry eggs enclosed in a sealed dorsal brood pouch. While most anurans are considered to be oviparous with lecithotrophic eggs, the extensively vascularized membrane of the brood pouch in marsupial frogs suggests potential opportunities for nutrient transfer. We tested for matrotrophy in the live-bearing Gastrotheca excubitor (Hemiphractidae), through feeding insects labelled with a 13C-fatty acid and a 15N-amino acid to brooding marsupial frogs. We observed significant increases of δ13C and δ15N in both maternal pouch tissues and embryos, suggesting nutrient transfer. Embryo dry mass also increased with developmental stage, providing further direct evidence for matrotrophy. These results suggest that in addition to gas exchange, the vascularized brood pouch membrane of G. excubitor also enables maternal nutrient transfer. This finding revealed a suspected but untested trait in the evolution of parental care in marsupial frogs, in contrast to previous work on Gastrotheca species that release tadpoles, and suggests greater complexity in reproductive and provisioning modes than previously thought.