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.     

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 Last South American Redoubt? Tested Surinamese Anurans Still Chytrid Free

Batrachochytrium dendrobatidis, a chytrid fungus infecting amphibians’ cutaneous layer, is responsible for the greatest contemporary loss of amphibian biodiversity. In South America, Suriname is one of the only three countries where B. dendrobatidis infections of anurans (frogs and toads) have not been documented. To further examine this apparent gap in pathogen occurrence, frogs were sampled for B. dendrobatidis spores at eight disparate geographic locations in Suriname, including locations with high and low levels of anthropogenic activities, and near Suriname’s border with Brazil and French Guiana, countries where B. dendrobatidis infections have been documented. None of the 347 frogs sampled, representing 37 species from eight families, tested positive for B. dendrobatidis. Our results provide the baseline data for future comparative testing and one of the last opportunities for a country in South America to proactively plan mitigation measures to protect amphibians from B. dendrobatidis’ presumed eventual incursion into Suriname.

     

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.     

<Emphasis Type="Italic">Batrachochytrium</Emphasis><Emphasis Type="Italic">dendrobatidis</Emphasis> and Chytridiomycosis in Anuran Amphibians of Colombia

In order to investigate the possible presence of the chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycota: Chytridiales) in frogs (Amphibia: Anura) of Colombia, we made a retrospective examination of formalin-fixed specimens preserved in natural history collections. Using the staining technique of hematoxylin and eosin to identify B. dendrobatidis in histological slices, we found evidence of the fungus in 3 of the 53 frog species examined from a total of 672 specimens collected in 17 departments within Colombia between 1968 and 2006. The infected specimens were found dead or dying in recent years in high elevation sites, suggesting that chytridiomycosis (the disease caused by the fungus) may represent a significant threat to Colombian amphibians. We conclude that a more extensive search for B. dendrobatidis in museum specimens and wild-caught frogs should be undertaken as soon as possible, using both histological and molecular genetic techniques, in order to further characterize the geographic and taxonomic extent of infections of B. dendrobatidis.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.     

Chytridiomycosis Widespread in Anurans of Northeastern United States

ABSTRACT An emerging disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis has been associated with morbidity, mortality, and extinction of species. Typically, researchers have detected B. dendrobatidis only when examining amphibians for causes of mortalities; few data exist on infection rates where mortalities are lacking. During May-September 2000–2002 we obtained amphibian specimens killed by vehicles and others collected at remote off-road sites throughout Maine, USA, and from federal lands in 5 states in the Northeast. We detected infected specimens, mostly green frogs (Rana clamitans), at 5 of 7 national wildlife refuges, a federal waterfowl production area, and Acadia National Park. Seven of 9 species, including all Ranidae species, were infected throughout Maine; rates ranged from 14.6% in American toads (Bufo americanus) to 25.7% in northern leopard frogs (Rana pipiens). We did not detect any infections in 50 eastern gray tree frogs (Hyla versicolor) or 21 spring peepers (Pseudacris crucifer). Species that hibernate in terrestrial habitats seem to have lower rates of infection than species that hibernate in aquatic habitats. Infections peaked in spring and autumn and were associated with air temperatures optimal for B. dendrobatidis growth. The relatively high infection rates among species without documented die-offs suggest that either losses have occurred undetected, that the fungus is endemic and species have attained a level of resistance to infections becoming lethal, or that climatic conditions of the Northeast have a role in preventing infections from being lethal. Data on prevalence and distribution of this chytrid fungus in the Northeast may be useful in modeling its origins and predicting long-term ecosystem effects involving anurans.     

How Does Chytrid Infection Vary Among Habitats? The Case of Litoria wilcoxii (Anura, Hylidae) in SE Queensland, Australia

Most analyses dealing with the geographical distribution of the chytrid fungus (Batrachochytrium dendrobatidis) have been performed on large geographical scales and data on more localized distribution of the chytrid within catchments are scarce. In this study, we compare the prevalence and intensity of infection of chytrid within and outside rainforest habitats at five independent catchments in southeast Queensland. In each catchment, we sampled adult Litoria wilcoxii along two transects on the same stream: one in forested areas, and the other in open nearby farmland. We analyzed swabs using quantitative PCR techniques. Male frogs were in higher densities in open habitats compared with the nearby forested areas. Infected male frogs were found in all catchments surveyed; however, prevalence of B. dendrobatidis in adult males was higher in the forested habitats than in the open habitats in four of the catchments. There was no significant difference in intensity of infection between forested and open habitats. For adult females and juveniles, sample sizes were not high enough for comparisons. Our results suggest that habitat influences chytrid prevalence and open areas may provide refuge from chytrid-induced population declines.     

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.     

Chytridiomycosis in Wild Frogs from Pico Bonito National Park, Honduras

Almost half of the endemic species of Honduran amphibians have declining populations; some of which seem to be extinct since they have not been seen in several years in places where they were once common. Disappearances in pristine and protected habitats have occurred in several highland localities throughout the country. The highland amphibian fauna of Pico Bonito National Park declined sometime between 1989 and 1995. An amphibian chytrid fungus, Batrachochytrium dendrobatidis has been linked to similar declines in other neotropical regions. We checked 19 specimens for this disease, which were collected in the park in 2003. The only Rana maculata examined was found to be infected, as were three of the six Eleutherodactylus aurilegulus surveyed for the disease. Two of the infected E. aurilegulus were collected at 120 m elevation and showed strong infections. One of these was lethargic and did not react when it was collected in the field, although it was still alive. A complete necropsy could help determine if the B. dendrobatidis infection was responsible for these symptoms, and further research might show how susceptible E. aurilegulus is to this pathogen at low altitudes. More research should be focused on the distribution of this pathogen in Honduras, and on how this disease has affected the local amphibian fauna.     

Amphibian Chytrid Fungus Broadly Distributed in the Brazilian Atlantic Rain Forest

To investigate the occurrence of the chytrid fungus Batrachochytrium dendrobatidis in Brazil, we conducted histological screenings of 96 preserved specimens of anurans collected at 10 sites in the Atlantic rain forest. Data show this fungus to be widely distributed. Infected specimens included Colostethus olfersioides (Dendrobatidae), Bokermannohyla gouveai and Hypsiboas freicanecae (Hylidae), as well as Thoropa miliaris and Crossodactylus caramaschii (Leptodactylidae), extending the area of B. dendrobatidis occurrence in Brazil approximately 1,600 km N, 200 km S, and 270 km E. The altitudinal range of the chytrid is broad, spanning from less than 100 m (Estação Ecológica Juréia-Itatins, Reserva Biológica do Tinguá) to about 2,400 m (Parque Nacional do Itatiaia). An infection record dating to 1981 roughly coincides with the time of the first observations of amphibian declines in the country. Widespread occurrence of B. dendrobatidis in the Atlantic Forest adds to the challenge of conserving an already endangered biome given the potential risk of further local biodiversity loss. Further research is needed to understand how environmental and genetic factors relate to chytridiomycosis in leading to or preventing local die-offs. Protected sites at mid and high elevations may be particularly threatened, while lowland populations may be functioning as reservoirs. Conservation efforts should also involve monitoring studies and habitat protection.     

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.     

Population Recovery following Decline in an Endangered Stream-Breeding Frog (Mixophyes fleayi) from Subtropical Australia

Amphibians have undergone dramatic declines and extinctions worldwide. Prominent among these have been the stream-breeding frogs in the rainforests of eastern Australia. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been postulated as the primary cause of these declines. We conducted a capture-mark-recapture study over a 7-year period on the endangered Fleay’s barred frog (Mixophyes fleayi) at two independent streams (30 km apart) in order to assess the stability of these populations. This species had undergone a severe decline across its narrow geographic range. Mark-recapture modelling showed that the number of individuals increased 3–10 fold along stream transects over this period. Frog detection probabilities were frequently above 50% but declined as the populations increased. Adult survival was important to overall population persistence in light of low recruitment events, suggesting that longevity may be a key factor in this recovery. One male and female were present in the capture record for >6 years. This study provides an unambiguous example of population recovery in the presence of Bd.     

Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs

Predicting how climate change will affect disease dynamics requires an understanding of how the environment affects host–pathogen interactions. For amphibians, global declines and extinctions have been linked to a pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Using a combination of body temperature measurements and disease assays conducted before and after the arrival of B. dendrobatidis, this study tested the hypothesis that body temperature affects the prevalence of infection in a wild population of Panamanian golden frogs (Atelopus zeteki). The timing of first detection of the fungus was consistent with that of a wave of epidemic infections spreading south and eastward through Central America. During the epidemic, many golden frogs modified their thermoregulatory behaviour, raising body temperatures above their normal set point. Odds of infection decreased with increasing body temperature, demonstrating that even slight environmental or behavioural changes have the potential to affect an individual''s vulnerability to infection. The thermal dependency of the relationship between B. dendrobatidis and its amphibian hosts demonstrates how the progression of an epidemic can be influenced by complex interactions between host and pathogen phenotypes and the environments in which they are found.     

Temporal Variation in Infection Prevalence by the Amphibian Chytrid Fungus in Three Species of Frogs at La Selva,Costa Rica

The emerging infectious disease chytridiomycosis, caused by the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is implicated in widespread population declines, extirpations, and extinctions of amphibians throughout the world. In the Neotropics, most amphibian declines have occurred in cool mid‐ to high‐elevation sites (> 400 m asl), and it is hypothesized that high temperatures limit the growth of Bd in lowland tropical sites, despite few data available on the distribution of Bd in lowland forests. Here, we report the results of a 12‐mo pathogen surveillance program for three common species of frogs at a warm lowland site in northeastern Costa Rica. We combine standard non‐invasive skin swabbing techniques with a quantitative polymerase chain reaction assay to analyze the infection prevalence and Bd load across a 1‐yr period. Our data indicate an overall Bd infection rate of 6.1 percent, but prevalence varies from < 5 percent in warmer months to a peak of 34.7 percent in the coolest months of the year. Despite very little seasonal variation in temperature (< 4°C), our data indicate strong seasonal variation in the prevalence of Bd, with highest prevalence of infection in months with coolest air temperatures. While it has been suggested that Bd is primarily a riparian fungus, we find no difference in prevalence of infection among our species despite considerable differences in affiliation of these species with water. Our study provides further evidence that infection by Bd is regulated by temperature and shows that warm temperatures in lowland forests may restrict, but not prevent, infection by Bd.     

Widespread triazole pesticide use affects infection dynamics of a global amphibian pathogen

The sixth mass extinction is a consequence of complex interplay between multiple stressors with negative impact on biodiversity. We here examine the interaction between two globally widespread anthropogenic drivers of amphibian declines: the fungal disease chytridiomycosis and antifungal use in agriculture. Field monitoring of 26 amphibian ponds in an agricultural landscape shows widespread occurrence of triazole fungicides in the water column throughout the amphibian breeding season, together with a negative correlation between early season application of epoxiconazole and the prevalence of chytrid infections in aquatic newts. While triazole concentrations in the ponds remained below those that inhibit growth of Batrachochytrium dendrobatidis, they bioaccumulated in the newts' skin up to tenfold, resulting in cutaneous growth-suppressing concentrations. As such, a concentration of epoxiconazole, 10 times below that needed to inhibit fungal growth, prevented chytrid infection in anuran tadpoles. The widespread presence of triazoles may thus alter chytrid dynamics in agricultural landscapes.     

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.