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.     

Motile Zoospores of Batrachochytrium dendrobatidis Move Away from Antifungal Metabolites Produced by Amphibian Skin Bacteria

Chytridiomycosis is an amphibian skin disease that threatens amphibian biodiversity worldwide. The fungal agent of chytridiomycosis is Batrachochytrium dendrobatidis. There is considerable variation in disease outcomes such that some individuals and populations co-exist with the fungus and others quickly succumb to disease. Amphibians in populations that co-exist with the B. dendrobatidis have sublethal infections on their skins. Symbiotic skin bacteria have been shown in experiments and surveys to play a role in protecting amphibians from chytridiomycosis. Little is known about the mechanisms that antifungal skin bacteria use to ameliorate the effects of B. dendrobatidis. In this study, we identified that B. dendrobatidis isolate JEL 310 zoospores display chemotaxis, in the presence of two bacterially-produced metabolites (2,4-diacetylphloroglucinol and indole-3-carboxaldehyde). In the presence of either metabolite, B. dendrobatidis zoospores move more frequently away from the metabolite. Using parameters estimated from this study, a simple stochastic model of a random walk on a lattice was evaluated. The model shows that these individual behaviors over short time-scales directly lead to population behaviors over long time–scales, such that most zoospores will escape, or not infect a tryptone substrate containing the bacterially-produced metabolite, whereas many zoospores will infect the tryptone substrate containing no metabolite. These results suggest that amphibians that have skin bacteria produce antifungal metabolites that might be able to keep B. dendrobatidis infections below the lethal threshold and thus are able to co-exist with the fungus.     

Amphibian chytridiomycosis in Japan: distribution,haplotypes and possible route of entry into Japan

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the first report of chytridiomycosis in Asia. To assess the risk of pandemic chytridiomycosis to Japanese frogs, we surveyed the distribution of the fungus among captive and wild frog populations. We established a nested PCR assay that uses two pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B. dendrobatidis DNA. We collected swab samples from 265 amphibians sold at pet shops, 294 bred at institutes and 2103 collected at field sites from northern to southwestern Japan. We detected infections in native and exotic species, both in captivity and in the field. Sequencing of PCR products revealed 26 haplotypes of the B. dendrobatidis ITS region. Phylogenetic analysis showed that three of these haplotypes were specific to the Japanese giant salamander (Andrias japonicus) and appeared to have established a commensal relationship with this native amphibian. Many other haplotypes were carried by alien amphibians. The highest genetic diversity of B. dendrobatidis was found in the American bullfrog (Rana catesbeiana). Some strains of B. dendrobatidis appeared to be endemic to Japanese native amphibians, but many alien strains are being introduced into Japan via imported amphibians. To improve chytridiomycosis risk management, we must consider the risk of B. dendrobatidis changing hosts as a result of anthropogenic disturbance of the host‐specific distribution of the fungus.     

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.     

Presence of the Chytrid Fungus Batrachochytrium dendrobatidis in Populations of the Critically Endangered Frog Mannophryne olmonae in Tobago, West Indies

The emerging infectious disease chytridiomycosis is prevalent in Central and South America, and has caused catastrophic declines of amphibian populations in the Neotropics. The responsible organism, Batrachochytrium dendrobatidis, has been recorded on three West Indian islands, but the whole of the Caribbean region is predicted to offer a suitable environment for the disease. Monitoring the spread of chytridiomycosis is thus a priority in this region, which has exceptionally high levels of amphibian endemism. PCR analysis of 124 amphibian skin swabs in Tobago (Republic of Trinidad and Tobago) demonstrated the presence of B. dendrobatidis in three widely separated populations of the frog Mannophryne olmonae, which is listed as Critically Endangered on the basis of recent population declines. Chytridiomycosis is presently endemic in this species, with a prevalence of about 20% and no associated clinical disease. Increased susceptibility to chytridiomycosis from climate change is unlikely in amphibian populations in Tobago, as this island does not have high montane environments, but remains a possibility in the sister island of Trinidad. Preventing the spread of chytridiomycosis within and between these and other Caribbean islands should be a major goal of practical conservation measures for amphibians in the region.     

Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines

Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 μM. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis.     

Ubiquity of the Pathogenic Chytrid Fungus, <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis>, in Anuran Communities in Panamá

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis, has been implicated as the main driver of many enigmatic amphibian declines in neotropical sites at high elevation. Batrachochytrium dendrobatidis is thought to be a waterborne pathogen limited by temperature, and the extent to which it persists and causes disease in amphibians at lower elevations in the neotropics is not known. It also is unclear by what mechanism(s) B. dendrobatidis has emerged as a pathogenic organism. To test whether B. dendrobatidis is limited by elevation in Panamá, we sought to determine the prevalence and intensity of B. dendrobatidis in relation to anuran abundance and diversity using quantitative PCR (qPCR) analyses. Sites were situated at varying elevations, from 45 to 1215 m, and were at varying stages of epizootic amphibian decline, including pre-epizootic, mid-epizootic, 2 years post-epizootic, and 10 years post-epizootic. Batrachochytrium dendrobatidis was found in all sites regardless of elevation or stage of epizootic decline. Levels of prevalence and infection intensity were comparable across all sites except at the mid-epizootic site, where both prevalence and intensity were significantly higher than at other sites. Symptoms of chytridiomycosis and corresponding declines in amphibian populations were variably seen at all elevations along a post-epizootic gradient. Because it is inherently difficult to prove a negative proposition, it can neither be proven that B. dendrobatidis is truly not present where it is not detected nor proven that it is only recently arrived where it is detected. Thus, there will always be doubts about whether B. dendrobatidis is enzootic or invasive. In any case, our results, coupled with current knowledge, suggest most clearly that the disease, chytridiomycosis, may be novel and invasive, and that the pathogen, B. dendrobatidis either is, or is becoming, globally ubiquitous.     

Distribution models for the amphibian chytrid Batrachochytrium dendrobatidis in Costa Rica: proposing climatic refuges as a conservation tool

Aim We use novel data on the occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in Costa Rica to model its potential distribution in that country. Location Lowland and montane areas of Costa Rica. Methods We use published and new data on the presence of B. dendrobatidis on 647 amphibians (35 species). Screening was performed through histological techniques by which 156 sites were surveyed. Of these, 21 were found to have the amphibian chytrid. Maxent, a presence‐only distribution modelling technique, was used to create 100 predictions of B. dendrobatidis occurrence, of which the most accurate 10 (based on area under the receiver‐operating characteristic curve) were chosen to create a composite distribution model. This approach increased confidence in model predictions, distinguishing areas of high probability of occurrence and low variability across model runs (higher confidence) from those with high probability but high variability (lower confidence). Results Predicted distribution patterns were not uniform along Costa Rica's mountains, where most amphibian declines have occurred. The pathogen was predicted to occur with greater probability on the Caribbean slopes than on the Pacific slopes. While high temperature seems to constrain the distribution of the pathogen, areas that also have small amounts of rainfall during the driest period of the year were predicted to have low probability of B. dendrobatidis occurrence. Main conclusions The model predicts that the Santa Elena Peninsula and the Central Valley have low probabilities of B. dendrobatidis occurrence, suggesting that they could function as refuges for amphibians. In such refugial areas, one could expect B. dendrobatidis to be absent, or to be present in low abundance (rendering an epidemic outbreak of chytridiomycosis unlikely). Craugastor ranoides, which belongs to a group of frogs particularly sensitive to chytridiomycosis outbreaks, persists in the hot and seasonally dry Santa Elena Peninsula but disappeared in the nearby colder and more humid Guanacaste Volcanic Chain. This information suggests that climatic refuges, where environmental conditions prevent disease outbreaks, could be an important component in amphibian conservation.     

Two novel antimicrobial peptides from skin secretions of the frog,Rana nigrovittata

Two novel antimicrobial peptides with similarity to brevinin‐2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin‐2‐RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin‐2‐RN2), respectively, by Edman degradation. Different from brevinin‐2, which is composed of 33 amino acid residues (aa), both brevinin‐2‐RN1 and ‐RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465‐9) encoding precursors of brevinin‐2‐RN1 and ‐RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin‐2‐RN. Both brevinin‐2‐RN1 and ‐RN2 showed strong antimicrobial activities against gram‐positive and gram‐negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Cytotoxic peptides with insulin‐releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae)

Peptidomic analysis of norepinephrine‐stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host‐defense peptides differing by a single amino acid residue belonging to the brevinin‐1 family (brevinin‐1ITa and ‐1ITb), a peptide belonging to the temporin family (temporin‐ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine‐sulphoxide forms of brevinin‐1ITa and ‐1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin‐1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non‐small cell lung adenocarcinoma A549 cells (LC₅₀ = 18 μM), breast adenocarcinoma MDA‐MB‐231 cells (LC₅₀ = 8 μM) and colorectal adenocarcinoma HT‐29 cells (LC₅₀ = 18 μM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC₅₀ = 7 μM). Temporin‐ITa (VFLGAIAQALTSLLGKL.NH₂) was between three and fivefold less potent against these cells. Brevinin‐1ITa inhibited growth of both Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin‐ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN‐BD11 clonal β‐cells at concentrations ≥1 nM, but brevinin‐1ITa was cytotoxic to the cells at concentrations ≥3 μM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Predation by zooplankton on <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis>: biological control of the deadly amphibian chytrid fungus?

Batrachochytrium dendrobatidis (hereafter Batrachochytrium), a fungal pathogen of amphibians, causes the disease chytridiomycosis which is responsible for unprecedented population declines and extinctions globally. Host defenses against chytridiomycosis include cutaneous symbiotic bacteria and anti-microbial peptides, and proposed treatment measures include use of fungicides and bioaugmentation. Efforts to eradicate the fungus from localized areas of disease outbreak have not been successful. Instead, control measures to mitigate the impacts of the disease on host populations, many of which are already persisting with Batrachochytrium in an endemic state, may be more realistic. The infective stage of the fungus is an aquatic zoospore, 3–5 μm in diameter. Here we show that zoospores of Batrachochytrium are consumed by the zooplankter Daphnia magna. This species inhabits amphibian breeding sites where Batrachochytrium transmission occurs, and consumption of Batrachochytrium zoospores may lead to effective biological control of Batrachochytrium.     

A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides

Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.     

The Amphibian Chytrid Batrachochytrium dendrobatidis Occurs on Freshwater Shrimp in Rain Forest Streams in Northern Queensland, Australia

Chytridiomycosis is a disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis. It can be highly virulent and is unusual in that it appears to drive many host species to local extinction during outbreaks. One mechanism that could facilitate this is the ability to grow saprophytically or on alternative hosts. This is common in other chytrids but has not been demonstrated for B. dendrobatidis in the field. B. dendrobatidis can grow on arthropod exoskeletons in the laboratory, and freshwater shrimp can be the most abundant animals in tropical rain forest streams. We therefore used diagnostic quantitative polymerase chain reaction to determine the infection status of freshwater shrimp from areas in which they are sympatric with frog species that have suffered declines in association with outbreaks of chytridiomycosis. We detected B. dendrobatidis on three individual shrimp belonging to two genera and collected from two widely separated streams. Two of the individuals had high levels of infection. This indicates that the presence of alternative hosts is likely to contribute to the extreme virulence of chytridiomycosis outbreaks in some systems. The presence of alternative hosts may allow B. dendrobatidis to remain in the environment after local extinctions of amphibian hosts, preventing the recovery of amphibian populations.     

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.     

The Bacterially Produced Metabolite Violacein Is Associated with Survival of Amphibians Infected with a Lethal Fungus

The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 μM on a salamander''s skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.The amphibian fungal pathogen Batrachochytrium dendrobatidis causes a lethal skin disease that has caused substantial declines in amphibian populations (18). However, some species, such as the bullfrog (Rana catesbeiana) and the tiger salamander (Ambystoma tigrinum), are relatively asymptomatic when they are infected with this pathogen (4, 5). Variation in survival among species has been attributed to differences in innate immune factors, such as antimicrobial peptides (20) and skin-associated microbial species (8-11), as well as behavior (16). The presence of antifungal microbes is of particular interest because it suggests that these organisms are mutualistic associates of amphibian species. In addition, augmentation of the cutaneous microbial community by adding species of bacteria that inhibit B. dendrobatidis has the potential to provide resistance to chytridiomycosis (9).We have identified a number of bacteria associated with the skin of amphibians that inhibit B. dendrobatidis in vitro via secretion of antifungal metabolites (2, 3, 10, 11). The bacterial species used in this study, Janthinobacterium lividum, produces the anti-B. dendrobatidis metabolites violacein and indole-3-carboxaldehyde (MIC, 1.82 μM and 69 μM, respectively) (3). We have shown that violacein inhibits B. dendrobatidis in laboratory assays (3) and is strongly correlated with survival in vivo of the frog species Rana muscosa (9). Violacein was also present on three of seven wild-collected red-backed salamanders (Plethodon cinereus) at concentrations that inhibit B. dendrobatidis in vitro (3), suggesting that this salamander species has a mutualistic community of violacein-producing bacteria on its skin. In this study, we added J. lividum to salamander skins to generate a wide range of violacein concentrations in order to determine what concentration is needed to prevent mortality caused by chytridiomycosis in vivo.     

Immune evasion or avoidance: Fungal skin infection linked to reduced defence peptides in Australian green-eyed treefrogs, Litoria serrata

Many parasites and pathogens suppress host immunity to maintain infection or initiate disease. On the skin of many amphibians, defensive peptides are active against the fungus Batrachochytrium dendrobatidis (Bd), the causative agent of the emerging infectious disease chytridiomycosis. We tested the hypothesis that infection with the fungus may be linked to lower levels of defensive peptides. We sampled both ambient (or constitutive) skin peptides on the ventral surface immediately upon capture, and stored skin peptides induced from granular glands by norepinephrine administration of Australian green-eyed treefrogs, Litoria serrata. Upon capture, uninfected frogs expressed an array of antimicrobial peptides on their ventral surface, whereas infected frogs had reduced skin peptide expression. Expression of ambient skin peptides differed with infection status, and antimicrobial peptides maculatin 1.1 and 2.1 were on average three times lower on infected frogs. However, the repertoire of skin peptides stored in granular glands did not differ with infection status; on average equal quantities were recovered from infected and from uninfected frogs. Our results could have at least two causes: (1) frogs with reduced peptide expression are more likely to become infected; (2) Bd infection interferes with defence peptides by inhibiting release or causing selective degradation of peptides on the skin surface. Immune evasion therefore may contribute to the pathogenesis of chytridiomycosis and a mechanistic understanding of this fungal strategy may lead to improved methods of disease control.     

The relationship between the emergence of Batrachochytrium dendrobatidis,the international trade in amphibians and introduced amphibian species

Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid Batrachochytrium dendrobatidis. The disease has been associated with global amphibian declines and species extinctions, however the principle drivers that underly the emergence of chytridiomycosis remain unclear. Current evidence suggests that the world trade in amphibians is implicated in the emergence of chytridiomycosis. Here, we review the evidence that the amphibian trade is driving the emergence of chytridiomycosis by (1) spreading infected animals worldwide, (2) introducing non-native infected animals into naïve populations and (3) amplifying infection of amphibians by co-housing, followed by untreated discharge of infectious zoospores into water supplies. We conclude that the evidence that the amphibian trade is contributing to the spread of Batrachochytrium dendrobatidis is strong, and that specific actions are necessary to prevent the introduction of the pathogen into thus-far uninfected areas. Specifically, we recommend the development of national risk-abatement plans, focused on firstly preventing introduction of Bd into disease free areas, and secondly, decreasing the impact of the disease on populations that are currently infected.     

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay.In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.     

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen (Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity—declining amphibian populations.