Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Lymphocyte Inhibition by the Salamander-Killing Chytrid Fungus, Batrachochytrium salamandrivorans.

Amphibian populations have been declining around the world for more than five decades, and the losses continue. Although causes are complex, major contributors to these declines are two chytrid fungi, Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, which both cause the disease termed chytridiomycosis. Previously, we showed that B. dendrobatidis impedes amphibian defenses by directly inhibiting lymphocytes in vitro and in vivo by release of soluble metabolites, including kynurenine (KYN), methylthioadenosine (MTA), and spermidine (SPD). Here, we show that B. salamandrivorans cells and cell-free supernatants also inhibit amphibian lymphocytes as well as a human T cell line. As we have shown for B. dendrobatidis, high-performance liquid chromatography (HPLC) and mass spectrometry revealed that KYN, MTA, and SPD are key metabolites found in the B. salamandrivorans supernatants. Production of inhibitory factors by B. salamandrivorans is limited to mature zoosporangia and can occur over a range of temperatures between 16°C and 26°C. Taken together, these results suggest that both pathogenic Batrachochytrium fungi have evolved similar mechanisms to inhibit lymphocytes in order to evade clearance by the amphibian immune system.

Preparatory immunity: Seasonality of mucosal skin defences and Batrachochytrium infections in Southern leopard frogs.

Accurately predicting the impacts of climate change on wildlife health requires a deeper understanding of seasonal rhythms in host-pathogen interactions. The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), exhibits seasonality in incidence; however, the role that biological rhythms in host defences play in defining this pattern remains largely unknown. The aim of this study was to examine whether host immune and microbiome defences against Bd correspond with infection risk and seasonal fluctuations in temperature and humidity. Over the course of a year, five populations of Southern leopard frogs (Rana [Lithobates] sphenocephala) in Tennessee, United States, were surveyed for host immunity, microbiome and pathogen dynamics. Frogs were swabbed for pathogen load and skin bacterial diversity and stimulated to release stored antimicrobial peptides (AMPs). Secretions were analysed to estimate total hydrophobic peptide concentrations, presence of known AMPs and effectiveness of Bd growth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibit Bd growth in vitro were used as an estimate of predicted anti-Bd function of the skin microbiome. Batrachochytrium dendrobatidis dynamics followed the expected seasonal fluctuations-peaks in cooler months-which coincided with when host mucosal defences were most potent against Bd. Specifically, the concentration and expression of stored AMPs cycled synchronously with Bd dynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibit Bd growth was greatest when risk of Bd infection was highest. We interpret the increase in peptide storage in the fall and the shift to a more anti-Bd microbiome over winter as a preparatory response for subsequent infection risk during the colder periods when AMP synthesis and bacterial growth is slow and pathogen pressure from this cool-adapted fungus is high. Given that a decrease in stored AMP concentrations as temperatures warm in spring likely means greater secretion rates, the subsequent decrease in prevalence suggests seasonality of Bd in this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression.

Emerging fungal pathogens pose a greater threat to biodiversity than any other parasitic group, causing declines of many taxa, including bats, corals, bees, snakes and amphibians. Currently, there is little evidence that wild animals can acquire resistance to these pathogens. Batrachochytrium dendrobatidis is a pathogenic fungus implicated in the recent global decline of amphibians. Here we demonstrate that three species of amphibians can acquire behavioural or immunological resistance to B. dendrobatidis. Frogs learned to avoid the fungus after just one B. dendrobatidis exposure and temperature-induced clearance. In subsequent experiments in which B. dendrobatidis avoidance was prevented, the number of previous exposures was a negative predictor of B. dendrobatidis burden on frogs and B. dendrobatidis-induced mortality, and was a positive predictor of lymphocyte abundance and proliferation. These results suggest that amphibians can acquire immunity to B. dendrobatidis that overcomes pathogen-induced immunosuppression and increases their survival. Importantly, exposure to dead fungus induced a similar magnitude of acquired resistance as exposure to live fungus. Exposure of frogs to B. dendrobatidis antigens might offer a practical way to protect pathogen-naive amphibians and facilitate the reintroduction of amphibians to locations in the wild where B. dendrobatidis persists. Moreover, given the conserved nature of vertebrate immune responses to fungi and the fact that many animals are capable of learning to avoid natural enemies, these results offer hope that other wild animal taxa threatened by invasive fungi might be rescued by management approaches based on herd immunity.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.

Out in the cold and sick: low temperatures and fungal infections impair a frog's skin defenses.

Amphibians worldwide continue to battle an emerging infectious disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Southern leopard frogs, Rana sphenocephala, are known to become infected with this pathogen, yet they are considered 'of least concern' for declines due to chytridiomycosis. Previous studies have shown that R. sphenocephala secretes four antimicrobial peptides (AMPs) onto their skin which may play an important role in limiting susceptibility to chytridiomycosis. Here, we examined (1) the effects of temperature and AMP depletion on infections with Bd and (2) the effects of temperature and Bd infection on the capacity to secrete AMPs in juvenile leopard frogs. Pathogen burden and mortality were greater in frogs exposed to Bd at low temperature but did not increase following monthly AMP depletion. Both low temperature and Bd exposure reduced the capacity of juvenile frogs to restore peptides after monthly depletions. Frogs held at 14°C were poorly able to restore peptides in comparison with those at 26°C. Frogs held at 26°C were better able to restore their peptides, but when exposed to Bd, this capacity was significantly reduced. These results strongly support the hypothesis that both colder temperatures and Bd infection impair the capacity of juvenile frogs to produce and secrete AMPs, an important component of their innate defense against chytrid fungi and other pathogens. Thus, in the face of unpredictable climate changes and enzootic pathogens, assessments of disease risk should consider the potential for effects of environmental variation and pathogen exposure on the quality of host defenses.

Frogs adapt to physiologically costly anthropogenic noise.

Human activities impose novel pressures on amphibians, which are experiencing unprecedented global declines, yet population-level responses are poorly understood. A growing body of literature has revealed that noise is an anthropogenic stressor that impacts ecological processes spanning subcellular to ecosystem levels. These consequences can impose novel selective pressures on populations, yet whether populations can adapt to noise is unknown. We tested for adaptation to traffic noise, a widespread sensory 'pollutant'. We collected eggs of wood frogs (Rana sylvatica) from populations from different traffic noise regimes, reared hatchlings under the same conditions, and tested frogs for differences in sublethal fitness-relevant effects of noise. We show that prolonged noise impaired production of antimicrobial peptides associated with defence against disease. Additionally, noise and origin site interacted to impact immune and stress responses. Noise exposure altered leucocyte production and increased baseline levels of the stress-relevant glucocorticoid, corticosterone, in frogs from quiet sites, but noise-legacy populations were unaffected. These results suggest noise-legacy populations have adapted to avoid fitness-relevant physiological costs of traffic noise. These findings advance our understanding of the consequences of novel soundscapes and reveal a pathway by which anthropogenic disturbance can enable adaptation to novel environments.

Immunomodulatory metabolites released by the frog-killing fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.

Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis.

The introduction of next-generation sequencing has allowed for greater understanding of community composition of symbiotic microbial communities. However, determining the function of individual members of these microbial communities still largely relies on culture-based methods. Here, we present results on the phylogenetic distribution of a defensive functional trait of cultured symbiotic bacteria associated with amphibians. Amphibians are host to a diverse community of cutaneous bacteria and some of these bacteria protect their host from the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) by secreting antifungal metabolites. We cultured over 450 bacterial isolates from the skins of Panamanian amphibian species and tested their interactions with Bd using an in vitro challenge assay. For a subset of isolates, we also completed coculture experiments and found that culturing isolates with Bd had no effect on inhibitory properties of the bacteria, but it significantly decreased metabolite secretion. In challenge assays, approximately 75% of the bacterial isolates inhibited Bd to some extent and these inhibitory isolates were widely distributed among all bacterial phyla. Although there was no clear phylogenetic signal of inhibition, three genera, Stenotrophomonas, Aeromonas and Pseudomonas, had a high proportion of inhibitory isolates (100%, 77% and 73%, respectively). Overall, our results demonstrate that antifungal properties are phylogenetically widespread in symbiotic microbial communities of Panamanian amphibians and that some functional redundancy for fungal inhibition occurs in these communities. We hope that these findings contribute to the discovery and development of probiotics for amphibians that can mitigate the threat of chytridiomycosis.

Effect of glucocorticoids on expression of cutaneous antimicrobial peptides in northern leopard frogs (Lithobates pipiens).

BACKGROUND: Many species of frogs secrete cutaneous antimicrobial peptides that are capable of killing Batrachochytrium dendrobatidis. Some of these species are nonetheless susceptible to chytridiomycosis, suggesting that host factors causing dysregulation of this innate immune response may be important in pathogenesis. Since stresses, such as from environmental perturbations, are a potential cause of such dysregulation, this study investigated the effect of glucocorticoid on cutaneous gene expression of these antimicrobial peptides. RESULTS: Northern leopard frogs (Lithobates pipiens) were injected with either the corticosteroid methylprednisolone or saline every 48 h. Norepinephrine-elicited cutaneous secretions were collected every 8 days for 40 days. Gene expression of antimicrobial peptides (brevinin-1P and ranatuerin-2P) in the cutaneous secretions was measured relative to the reference genes EF1-α and RPL8 using quantitative RT-PCR. Corticosteroid treatment was associated with a significant increase in brevinin-1P gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin-2P expression followed a similar but non-significant trend. CONCLUSION: This treatment protocol, including corticosteroid-administration and frequent norepinephrine-induced secretion, increased AMP gene expression in the skin of L. pipiens under these experimental conditions. The findings do not support the hypothesis that environmental stress predisposes frogs to chytridiomycosis by causing glucocorticoid-induced suppression of antimicrobial peptide defences.

Coqui frogs persist with the deadly chytrid fungus despite a lack of defensive antimicrobial peptides.

The amphibian skin fungus Batrachochytrium dendrobatidis (Bd) occurs widely in Puerto Rico and is thought to be responsible for the apparent extinction of 3 species of endemic frogs in the genus Eleutherodactylus, known as coquis. To examine immune defenses which may protect surviving species, we induced secretion of skin peptides from adult common coqui frogs E. coqui collected from upland forests at El Yunque. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we were unable to detect peptide signals suggestive of antimicrobial peptides, and enriched peptides showed no capacity to inhibit growth of Bd. Thus, it appears that E. coqui depend on other skin defenses to survive in the presence of this deadly fungus.

Inhibition of local immune responses by the frog-killing fungus Batrachochytrium dendrobatidis.

Amphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.

Evaluation of amphotericin B and chloramphenicol as alternative drugs for treatment of chytridiomycosis and their impacts on innate skin defenses.

Chytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogen Batrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 µg/ml), amphotericin B (0.8 to 1.6 µg/ml), and itraconazole (Sporanox) (20 ng/ml) against B. dendrobatidis. Both chloramphenicol and amphotericin B significantly reduced B. dendrobatidis infection in naturally infected southern leopard frogs (Rana [Lithobates] sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure of R. sphenocephala to these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multiple R. sphenocephala skin bacterial isolates in vitro at concentrations below the MIC against B. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis.

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis.

One issue of great concern for the scientific community is the continuing loss of diverse amphibian species on a global scale. Amphibian populations around the world are experiencing serious losses due to the chytrid fungus, Batrachochytrium dendrobatidis. This pathogen colonizes the skin, leading to the disruption of ionic balance and eventual cardiac arrest. In many species, antimicrobial peptides secreted into the mucus are thought to contribute to protection against colonization by skin pathogens. Although it is generally thought that antimicrobial peptides are an important component of innate immune defenses against B. dendrobatidis, much of the current evidence relies on correlations between effective antimicrobial peptide defenses and species survival. There have been few studies to directly demonstrate that antimicrobial peptides play a role. Using the northern leopard frog, Rana pipiens, we show here that injection of noradrenaline (norepinephrine) brings about a long-term depletion of skin peptides (initial concentrations do not recover until after day 56). When peptide stores recovered, the renewed peptides were similar in composition to the initial peptides as determined by MALDI-TOF mass spectrometry and in activity against B. dendrobatidis as determined by growth inhibition assays. Newly metamorphosed froglets depleted of their peptide stores and exposed to B. dendrobatidis died more rapidly than B. dendrobatidis-exposed froglets with their peptides intact. Thus, antimicrobial peptides in the skin mucus appear to provide some resistance to B. dendrobatidis infections, and it is important for biologists to recognize that this defense is especially important for newly metamorphosed frogs in which the adaptive immune system is still immature.

Larval exposure to predator cues alters immune function and response to a fungal pathogen in post-metamorphic wood frogs.

For the past several decades, amphibian populations have been decreasing around the globe at an unprecedented rate. Batrachochytrium dendrobatidis (Bd), the fungal pathogen that causes chytridiomycosis in amphibians, is contributing to amphibian declines. Natural and anthropogenic environmental factors are hypothesized to contribute to these declines by reducing the immunocompetence of amphibian hosts, making them more susceptible to infection. Antimicrobial peptides (AMPs) produced in the granular glands of a frog's skin are thought to be a key defense against Bd infection. These peptides may be a critical immune defense during metamorphosis because many acquired immune functions are suppressed during this time. To test if stressors alter AMP production and survival of frogs exposed to Bd, we exposed wood frog (Lithobates sylvaticus) tadpoles to the presence or absence of dragonfly predator cues crossed with a single exposure to three nominal concentrations of the insecticide malathion (0, 10, or 100 parts per billion [ppb]). We then exposed a subset of post-metamorphic frogs to the presence or absence of Bd zoospores and measured frog survival. Although predator cues and malathion had no effect on survival or size at metamorphosis, predator cues increased the time to metamorphosis by 1.5 days and caused a trend of a 20% decrease in hydrophobic skin peptides. Despite this decrease in peptides determined shortly after metamorphosis, previous exposure to predator cues increased survival in both Bd-exposed and unexposed frogs several weeks after metamorphosis. These results suggest that exposing tadpoles to predator cues confers fitness benefits later in life.

Evaluation of the skin peptide defenses of the Oregon spotted frog Rana pretiosa against infection by the chytrid fungus Batrachochytrium dendrobatidis.

Population declines due to amphibian chytridiomycosis among selected species of ranid frogs from western North America have been severe, but there is evidence that the Oregon spotted frog, Rana pretiosa Baird and Girard, 1853, displays resistance to the disease. Norepinephrine-stimulated skin secretions were collected from a non-declining population of R. pretiosa that had been exposed to the causative agent Batrachochytrium dendrobatidis. Peptidomic analysis led to identification and isolation, in pure form, of a total of 18 host-defense peptides that were characterized structurally. Brevinin-1PRa, -1PRb, -1PRc, and -1PRd, esculentin-2PRa and -PRb, ranatuerin-2PRa, -2PRb, -2PRc, and -2PRe, temporin-PRb and -PRc were identified in an earlier study of skin secretions of frogs from a different population of R. pretiosa known to be declining. Ranatuerin-2PRf, -2PRg, -2PRh, temporin-PRd, -PRe, and -PRf were not identified in skin secretions from frogs from the declining population, whereas temporin-PRa and ranatuerin-2PRd, present in skin secretions from the declining population, were not detected in the current study. All purified peptides inhibited the growth of B. dendrobatidis zoospores. Peptides of the brevinin-1 and esculentin-2 families displayed the highest potency (minimum inhibitory concentration = 6.25-12.5 µM). The study provides support for the hypothesis that the multiplicity and diversity of the antimicrobial peptide repertoire in R. pretiosa and the high growth-inhibitory potency of certain peptides against B. dendrobatidis are important in conferring a measure of resistance to fatal chytridiomycosis.

The importance of antimicrobial peptides (AMPs) in amphibian skin defense.

Antimicrobial peptides (AMPs) are produced for defense in nearly all taxa from simple bacteria to complex mammalian species. Some amphibian families have developed this defensive strategy to a high level of sophistication by loading the AMPs into specialized granular glands within the dermis. Enervated by the sympathetic nervous system, the granular glands are poised to deliver an array of AMPs to cleanse the wound and facilitate healing. There have been a number of excellent review publications in recent years that describe amphibian AMPs with an emphasis on their possible uses for human medicine. Instead, my aim here is to review what is known about the nature of amphibian AMPs, the diversity of amphibian AMPs, regulation of their production, and to provide the accumulated evidence that they do, indeed, play an important role in the protection of amphibian skin, vital for survival. While much has been learned about amphibian AMPs, there are still important gaps in our understanding of peptide synthesis, storage, and functions.

Heat stress and amphibian immunity in a time of climate change.

As a class of vertebrates, amphibians, are at greater risk for declines or extinctions than any other vertebrate group, including birds and mammals. There are many threats, including habitat destruction, invasive species, overuse by humans, toxic chemicals and emerging diseases. Climate change which brings unpredictable temperature changes and rainfall constitutes an additional threat. Survival of amphibians depends on immune defences functioning well under these combined threats. Here, we review the current state of knowledge of how amphibians respond to some natural stressors, including heat and desiccation stress, and the limited studies of the immune defences under these stressful conditions. In general, the current studies suggest that desiccation and heat stress can activate the hypothalamus pituitary-interrenal axis, with possible suppression of some innate and lymphocyte-mediated responses. Elevated temperatures can alter microbial communities in amphibian skin and gut, resulting in possible dysbiosis that fosters reduced resistance to pathogens. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.