The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines

Amphibian species have experienced population declines and extinctions worldwide that are unprecedented in recent history. Many of these recent declines have been linked to a pathogenic skin fungus, Batrachochytrium dendrobatidis, or to iridoviruses of the genus Ranavirus. One of the first lines of defense against pathogens that enter by way of the skin are antimicrobial peptides synthesized and stored in dermal granular glands and secreted into the mucus following alarm or injury. Here, I review what is known about the capacity of amphibian antimicrobial peptides from diverse amphibians to inhibit B. dendrobatidis or ranavirus infections. When multiple species were compared for the effectiveness of their in vitro antimicrobial peptides defenses against B. dendrobatidis, non-declining species of rainforest amphibians had more effective antimicrobial peptides than species in the same habitat that had recently experienced population declines. Further, there was a significant correlation between the effectiveness of the antimicrobial peptides and resistance of the species to experimental infection. These studies support the hypothesis that antimicrobial peptides are an important component of innate defenses against B. dendrobatidis. Some amphibian antimicrobial peptides inhibit ranavirus infections and infection of human T lymphocytes by the human immunodeficiency virus (HIV). An effective antimicrobial peptide defense against skin pathogens appears to depend on a diverse array of genes expressing antimicrobial peptides. The production of antimicrobial peptides may be regulated by signals from the pathogens. However, this defense must also accommodate potentially beneficial microbes on the skin that compete or inhibit growth of the pathogens. How this delicate balancing act is accomplished is an important area of future research.     

Predicted disease susceptibility in a Panamanian amphibian assemblage based on skin peptide defenses

Chytridiomycosis is an emerging infectious disease of amphibians caused by a chytrid fungus, Batrachochytrium dendrobatidis. This panzootic does not equally affect all amphibian species within an assemblage; some populations decline, others persist. Little is known about the factors that affect disease resistance. Differences in behavior, life history, biogeography, or immune function may impact survival. We found that an innate immune defense, antimicrobial skin peptides, varied significantly among species within a rainforest stream amphibian assemblage that has not been exposed to B. dendrobatidis. If exposed, all amphibian species at this central Panamanian site are at risk of population declines. In vitro pathogen growth inhibition by peptides from Panamanian species compared with species with known resistance (Rana pipiens and Xenopus laevis) or susceptibility (Bufo boreas) suggests that of the nine species examined, two species (Centrolene prosoblepon and Phyllomedusa lemur) may demonstrate strong resistance, and the other species will have a higher risk of disease-associated population declines. We found little variation among geographically distinct B. dendrobatidis isolates in sensitivity to an amphibian skin peptide mixture. This supports the hypothesis that B. dendrobatidis is a generalist pathogen and that species possessing an innate immunologic defense at the time of disease emergence are more likely to survive.     

Unlocking the story in the swab: A new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole‐genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field‐collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole‐genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Chytrid fungus parasitizing the wild amphibian Leptodactylus ocellatus (Anura: Leptodactylidae) in Argentina

The present contribution is the first report of parasitosis by a chytrid fungus in wild anuran amphibians in Argentina, as well as the first case of amphibian mortality documented to date in Argentina. We report the presence of the chytrid fungus in dead adult Leptodactylus ocellatus. It has been suggested that chytridiomycosis is the main cause of death in several amphibian populations worldwide. Our study demonstrates that chytridiomycosis afflicts L. ocellatus, a common widespread amphibian species, and is the first report of chytridiomycosis in the Argentinian lowlands. The occurrence at this latitude would indicate an extended distribution of this fungus in wildlife populations. It is also the first report of amphibian mortality due to chytrid fungus in our country. It is noteworthy that the site of collection is situated very close to sea level in a temperate climate zone and that this represents the southern most record for South American wild amphibians.     

There are abundant antimicrobial peptides in brains of two kinds of Bombina toads

It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin-H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 1-8, 10, 11, maximin H1, 3-5, 7, 9, 10, 12, 15, 16) from B. maxima skin secretions. Fifty-nine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and -negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains.     

Antimicrobial peptide defenses of the Tarahumara frog,Rana tarahumarae

Populations of the Tarahumara frog Rana tarahumarae have decreased markedly in recent years in the northern part of their range. Infection by the chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines. To determine whether antimicrobial peptides in the skin provide protection against this pathogen, norepinephrine-stimulated skin secretions were tested for their ability to inhibit growth of B. dendrobatidis in vitro. After concentration, crude mixtures of skin peptides inhibited the growth of the chytrid in a concentration-dependent manner. Proteomic analysis led to the identification and characterization of three peptides belonging to the brevinin-1 family of antimicrobial peptides and three belonging to the ranatuerin-2 family. The two most abundant peptides, ranatuerin-2TRa (GIMDSIKGAAKEIAGHLLDNLKCKITGC) and brevinin-1TRa (FLPVIAGIAANVLPKLFCKLTKRC), were active against B. dendrobatidis (MIC of 50 microM for ranatuerin-2TRa and 12.5 microM for brevinin-1TRa against zoospores). These data clearly show that antimicrobial peptides in the skin secretions of the Tarahumara frog are active against B. dendrobatidis and should provide some protection against infection. Therefore, the observed susceptibility of these frogs to this pathogen in the wild may be due to the effects of additional environmental factors that impair this innate defense mechanism, leading to the observed population declines.     

Using amphibians in laboratory studies: precautions against the emerging infectious disease chytridiomycosis

The African clawed frog Xenopus laevis is by far the most widely used amphibian species in laboratories. In the wild, X. laevis is an asymptomatic carrier of an emerging infectious disease called chytridiomycosis. The vector is the chytrid fungus Batrachochytrium dendrobatidis (Bd), which has devastating effects on wild amphibian populations around the world. The impact of Bd on the metabolism of X. laevis has not been comprehended yet. However, even if asymptomatic, an infection is likely to affect the individual's physiology, immunology, development, reproduction and overall response to stress from a purely medical point of view, which will introduce noise and therefore increase variance within experimental groups of X. laevis. This could have implications on the scientific results from studies using this species. Here, we review the current knowledge on treatments of infected amphibians and propose a hygiene protocol adapted to laboratory populations and amphibian husbandry. Following the presented sanitation guidelines could further prevent the spread of Bd and probably of other amphibian pathogens. The sanitation guidelines will help to reduce the impact of amphibian husbandry on natural populations and must be considered a crucial contribution to amphibian conservation, as today 32% of all amphibians are considered threatened.     

Ex situ Diet Influences the Bacterial Community Associated with the Skin of Red-Eyed Tree Frogs (Agalychnis callidryas)

Amphibians support symbiotic bacterial communities on their skin that protect against a range of infectious pathogens, including the amphibian chytrid fungus. The conditions under which amphibians are maintained in captivity (e.g. diet, substrate, enrichment) in ex situ conservation programmes may affect the composition of the bacterial community. In addition, ex situ amphibian populations may support different bacterial communities in comparison to in situ populations of the same species. This could have implications for the suitability of populations intended for reintroduction, as well as the success of probiotic bacterial inoculations intended to provide amphibians with a bacterial community that resists invasion by the chytrid fungus. We aimed to investigate the effect of a carotenoid-enriched diet on the culturable bacterial community associated with captive red-eyed tree frogs (Agalychnis callidryas) and make comparisons to bacteria isolated from a wild population from the Chiquibul Rainforest in Belize. We successfully showed carotenoid availability influences the overall community composition, species richness and abundance of the bacterial community associated with the skin of captive frogs, with A. callidryas fed a carotenoid-enriched diet supporting a greater species richness and abundance of bacteria than those fed a carotenoid-free diet. Our results suggest that availability of carotenoids in the diet of captive frogs is likely to be beneficial for the bacterial community associated with the skin. We also found wild A. callidryas hosted more than double the number of different bacterial species than captive frogs with very little commonality between species. This suggests frogs in captivity may support a reduced and diverged bacterial community in comparison to wild populations of the same species, which could have particular relevance for ex situ conservation projects.     

Adaptations of skin peptide defences and possible response to the amphibian chytrid fungus in populations of Australian green‐eyed treefrogs,Litoria genimaculata

Aim Rapidly evolving pathogens may exert diversifying selection on genes involved in host immune defence including those encoding antimicrobial peptides (AMPs). Amphibian skin peptides are one important defence against chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). We examined the population‐level variation in this innate immune defence to understand its relationship with disease dynamics. Location Queensland, Australia. Methods We examined skin peptide defences in five geographically distinct populations of Australian green‐eyed treefrogs, Litoria genimaculata. Skin peptide samples were collected from 52 frogs from three upland populations that previously declined as chytridiomycosis emerged, but subsequently recovered, and from 34 frogs in two lowland populations that did not decline. Historical samples of skin peptides preceding Bd emergence were not available from any population. Results In general, lowland populations had more effective peptide defences than upland populations. Peptide profiles were similar among populations, although relative amounts of peptides expressed differed significantly among populations and were more variable in the uplands. Infected frogs in upland populations carried a significantly higher infection burden compared to lowland populations. The presence of effective AMPs in the skin of L. genimaculata does not eliminate infection; however, more effective peptide defences may limit infection intensity and the progression of disease. Main conclusions The population bottleneck in upland populations caused by chytridiomycosis emergence did not appear to produce responses to selection for more effective peptide defences against chytridiomycosis compared to lowland populations of L. genimaculata. This does not exclude the possibility that current peptide defences have adapted in response to disease emergence. A suggestive (P < 0.10) interaction between infection status and population indicates that in lowland populations, infected individuals tend to be those with lower relative intensities of AMPs, whereas in the upland populations, infected and uninfected individuals are similar. Thus, both the AMPs and the environment may act to mediate resistance to Bd infection.     

The contribution of skin antimicrobial peptides to the system of innate immunity in anurans

Cationic peptides with the propensity to adopt an amphipathic ??-helical conformation in a membrane-mimetic environment are synthesized in the skins of many species of anurans (frogs and toads). These peptides frequently display cytolytic activities against a range of pathogenic bacteria and fungi consistent with the idea that they play a role in the host's system of innate immunity. However, the importance of the peptides in the survival strategy of the animal is not clearly understood. It is a common misconception that antimicrobial peptides are synthesized in the skins of all anurans. In fact, the species distribution is sporadic suggesting that their production may confer some evolutionary advantage to the organism but is not necessary for survival. Although growth inhibitory activity against the chytrid fungus Batrachochytrium dendrobatidis, responsible for anuran population declines worldwide, has been demonstrated in vitro, the ability of frog skin antimicrobial peptides to protect the animal in the wild appears to be limited and there is no clear correlation between their production by a species and its resistance to fatal chytridiomycosis. The low potency of many frog skin antimicrobial peptides is consistent with the hypothesis that cutaneous symbiotic bacteria may provide the major system of defense against pathogenic microorganisms in the environment with antimicrobial peptides assuming a supplementary role in some species.     

壶菌病与两栖动物的种群衰退

两栖类种群全球性衰退是21世纪最紧迫的环境问题之一。越来越多的证据表明壶菌(Batrachochytrium dendrobatidis)与澳大利亚、美洲北部、中部、南部及欧洲的两栖类种群衰退有关。由壶菌引起的壶菌病是变态后的无尾类所患的一种显性传染病,其令人质疑的快速传播及广泛爆发对世界范围内的两栖类种群构成重大威胁。本文对这种致病壶菌的病理学、生态学、生物地理学及其治疗方面的进展进行了综述。     

Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs

Many species of amphibians in the wet tropics of Australia have experienced population declines linked with the emergence of a skin-invasive chytrid fungus, Batrachochytrium dendrobatidis. An innate defense, antimicrobial peptides produced by granular glands in the skin, may protect some species from disease. Here we present evidence that supports this hypothesis. We tested ten synthesized peptides produced by Australian species, and natural peptide mixtures from five Queensland rainforest species. Natural mixtures and most peptides tested in isolation inhibited growth of B. dendrobatidis in vitro. The three most active peptides (caerin 1.9, maculatin 1.1, and caerin 1.1) were found in the secretions of non-declining species (Litoria chloris, L. caerulea, and L. genimaculata). Although the possession of a potent isolated antimicrobial peptide does not guarantee protection from infection, non-declining species (L. lesueuri and L. genimaculata) inhabiting the rainforest of Queensland possess mixtures of peptides that may be more protective than those of the species occurring in the same habitat that have recently experienced population declines associated with chytridiomycosis (L. nannotis, L. rheocola, and Nyctimystes dayi). This study demonstrates that in vitro effectiveness of skin peptides correlates with the degree of decline in the face of an emerging pathogen. Further research is needed to assess whether this non-specific immune defense may be useful in predicting disease susceptibility in other species.     

Anti-infection peptidomics of amphibian skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and "shuffling" of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and "host defense" properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further.     

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.     

Amphibian immune defenses against chytridiomycosis: impacts of changing environments

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.     

Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.     

The Link Between Rapid Enigmatic Amphibian Decline and the Globally Emerging Chytrid Fungus

Amphibians are globally declining and approximately one-third of all species are threatened with extinction. Some of the most severe declines have occurred suddenly and for unknown reasons in apparently pristine habitats. It has been hypothesized that these “rapid enigmatic declines” are the result of a panzootic of the disease chytridiomycosis caused by globally emerging amphibian chytrid fungus. In a Species Distribution Model, we identified the potential distribution of this pathogen. Areas and species from which rapid enigmatic decline are known significantly overlap with those of highest environmental suitability to the chytrid fungus. We confirm the plausibility of a link between rapid enigmatic decline in worldwide amphibian species and epizootic chytridiomycosis.