Chytrid Fungus Batrachochytrium Dendrobatidis Research Articles

Seasonal variation in the prevalence of a fungal pathogen and unexpected clearance from infection in a susceptible frog species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is a primary driver for amphibian population declines and extinctions worldwide. For highly susceptible species, such as the green and golden bell frog Litoria aurea, large numbers of Bd-related mortalities are thought to occur during the colder season (winter), when low temperatures favour the growth of the pathogen. However, extant L. aurea populations are persisting with Bd. We measured Bd prevalence and infection levels of wild L. aurea using capture-mark-recapture and radio-tracking methods. Using this information, we sought to determine host and environmental correlates of Bd prevalence and infection load. Mean ± SE infection load was higher in frogs sampled in autumn (431.5 ± 310.4 genomic equivalents; GE) and winter (1147.5 ± 735.8 GE), compared to spring (21.8 ± 19.3 GE) and summer (0.9 ± 0.8 GE). Furthermore, prevalence of Bd infection in L. aurea was highest in winter (43.6%; 95% CI 33.1-54.7%) and lowest in summer (11.2%; 95% CI 6.8-17.9%). Both prevalence and infection load decreased with increasing temperature. Seven frogs cleared their fungal infection during the coolest months when Bd prevalence was highest; however, these clearances were not permanent, as 5 frogs became infected again. Understanding the factors that allow amphibians to clear their Bd infections when temperatures are optimal for Bd growth presents the potential for manipulating such factors and provides an important step in future research.

Surveying for Batrachochytrium dendrobatidis and B. salamandrivorans in wild and captive amphibian populations in Estonia and Latvia.

The pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) cause infections that have become primary drivers of amphibian biodiversity loss. While globally widespread, the distribution margins of Bd and Bsal have not been determined, and the presence of these pathogens has probably gone unnoticed in many areas, especially in northern Eurasia. To better understand the presence and distribution of both pathogens in the northern temperate and boreal forest biomes, 243 individuals were sampled from 8 native amphibian species across Estonia. Additionally, 68 amphibians were sampled from captive collections in Estonia and Latvia. Pathogen infection was assessed using metabarcoding of the ITS2 marker. No positive matches for Bsal infection were found. Bd was detected in 13 specimens, 3 of which were sampled at the Riga Zoo (with a prevalence of 5.2%) and 10 in natural environments in Estonia (3.3%). The infected wild individuals belonged to 6 amphibian species and were detected throughout the mainland of Estonia, but not on islands. Prevalence of infection with Bd ranged between 3.1 and 12.5% among native species. In addition, we found molecular evidence for a potentially new sister species to Bd in nature. Although outbreaks of chytridiomycosis have never been observed in Estonia, it cannot be excluded that the dynamics of local amphibian populations are affected by Bd infections. Therefore, further work, including capture-mark-recapture studies and long-term monitoring, are required to clarify the impact of Bd on amphibians in Northern Europe.

Seasonal prevalence of the amphibian chytrid in a tropical pond-dwelling tadpole species.

Infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians and has caused catastrophic global declines of amphibian populations. Some studies have detected a seasonal pattern of infection associated with the local climate, and although most of them have focused on investigating the seasonality of Bd in relation to its impacts on amphibians, fewer have aimed to understand the chytrid persistence in the amphibian assemblage over seasons by investigating reservoir hosts. Since tadpoles are generally tolerant to Bd infection, they often play a relevant role in local disease dynamics. Thus, we hypothesized that tadpoles of Boana faber, a species that can be found in permanent ponds throughout the seasons, would function as Bd reservoirs. We therefore investigated Bd infection prevalence in tadpoles of this species over 2 yr in a nature reserve. As expected, we detected a seasonal variation of Bd infection, with a higher prevalence of Bd during the coldest months (winter) when compared to the warmer months (summer). Interestingly, our seasonal-trend decomposition analysis showed that Bd prevalence is increasing annually in the area, which could represent either a natural fluctuation of this pathogen, or an imminent threat to that anuran assemblage. With this study, we highlight the tadpole of B. faber as a potential reservoir for Bd, and we suggest that monitoring Bd in such hosts could be a powerful tool for identifying priority areas for amphibian conservation.

Locality, time and heterozygosity affect chytrid infection in yellow-bellied toads.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) infects numerous amphibian species worldwide and is suggested to drive population declines and extinction events. We report a study of Bd infection at the northernmost distribution of the European yellow-bellied toad Bombina variegata. A total of 577 individuals from ponds in 16 study sites were sampled for DNA and Bd throughout the breeding season. Microsatellite genotyping revealed 3 genetic clusters for the host B. variegata with an overall low genetic diversity. One of the clusters displayed a low microsatellite heterozygosity, a high inbreeding coefficient as well as high Bd infection prevalence and intensities. Multi-model estimates identified site, time of sampling, and heterozygosity to be important predictors of an individual's Bd infection status, and identified a strong effect of site on individual Bd infection intensity. The study site effects are suggestive of localized infection peaks, and the increase of individual Bd infection probabilities towards the end of the sampling period suggests cumulative infection during the breeding season. This study highlights the need for regular monitoring of Bd infection variables at multiple localities and times to gain insights into Bd dynamics. Due to the detected relationship between individual Bd infection status and heterozygosity, conservation measures should focus on the maintenance of high genetic diversity and connectivity within and among amphibian populations.

Apparent absence of the amphibian chytrid fungus (Batrachochytrium dendrobatidis) in frogs in Malaita Province, Solomon Islands

A major driver of global biodiversity loss is disease. One of the most devastating wildlife diseases known is chytridiomycosis, which is caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis, and is implicated in population declines in over 500 frog species. Thought to originate in Asia, B. dendrobatidis now has a global distribution, likely due to human movement and trade. The pathogen has yet to be detected in Melanesia, but there have been few surveys for B. dendrobatidis in the region, and none in the Solomon Islands archipelago, a biogeographic region with a unique and culturally important frog fauna. We swabbed 200 frogs of eight species in three genera in lowland and highland sites in East Kwaio on the island of Malaita in the Solomon Islands. All frogs tested negative for the pathogen but it is possible that the pathogen is present despite non-detection, so further surveys for the pathogen are needed throughout the country. Despite this, it is safest to take a precautionary approach and assume that B. dendrobatidis has not yet been introduced to the Solomon Islands, and that naïve native amphibian populations may be at risk of decline if the pathogen is introduced. Protocols are needed to prevent the accidental import of infected frogs via tourism or in logging or mining equipment. Monitoring of frog populations near areas of high risk such as ports is also recommended. The frogs of the Solomon Islands archipelago are biologically unique and culturally significant, and protecting them from the potentially devastating impacts of B. dendrobatidis is vital.

Instant killing of pathogenic chytrid fungi by disposable nitrile gloves prevents disease transmission between amphibians.

To prevent transmission of the pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), hygiene protocols prescribe the single use of disposable gloves for handling amphibians. We discovered that rinse water from nitrile gloves instantly kills 99% of Bd and Bsal zoospores. Transmission experiments using midwife toads (Alytes obstetricans) and Bd, and Alpine newts (Ichthyosaura alpestris) and Bsal, show that the use of the same pair of gloves for 2 subsequent individuals does not result in significant transmission of any chytrid fungus. In contrast, handling infected amphibians bare-handed caused transmission of Bsal in 4 out of 10 replicates, but did not result in transmission of Bd. Based on the manufacturer’s information, high resolution mass spectrometry (HRMS) and colorimetric tests, calcium lactate and calcium nitrate were identified as compounds with antifungal activity against both Bd and Bsal. These findings corroborate the importance of wearing gloves as an important sanitary measure in amphibian disease prevention. If the highly recommended single use of gloves is not possible, handling multiple post-metamorphic amphibians with the same pair of nitrile gloves should still be preferred above bare-handed manipulation.

Amphibian Infection Risk Changes with Host Life Stage and across a Landscape Gradient

Infectious pathogens threaten wildlife populations through effects on host growth, reproduction, and survival. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), the cause of the disease chytridiomycosis, has been implicated in worldwide declines of amphibian populations. Documenting conditions under which amphibians are threatened by Bd will allow us to pinpoint at-risk populations, especially in the midwestern United States, an understudied region. We investigated how distributions of Bd differ across host life stages, land cover types, and spatial extents in a susceptible temperate host, the American Toad (Anaxyrus americanus). We surveyed a total of 232 adults at 14 sites and 430 metamorphs at 15 sites in southwestern Ohio, USA, and analyzed associations among land use type, Bd prevalence, and Bd load by using model selection. We found Bd infection prevalence in metamorphs was dramatically lower than in adults; only 1.6% of metamorphs were infected compared with 28.0% of adults. These results suggest that Bd transmission occurs after metamorphosis in this species or that infections vary with season, given that we surveyed adults in the spring during breeding events and metamorphs in the summer as they emerged from ponds. In adults, infection prevalence was reduced with increasing open-canopy habitats across spatial scales (from 100 to 1,000 m), whereas infection load increased with the proportion of forested habitats at small spatial scales (100 m). Our study shows that Bd infection risk in a temperate system can be influenced by host life stage and land cover types across local spatial scales.

Species distribution models predict the geographic expansion of an enzootic amphibian pathogen

AbstractGlobally, numerous amphibian species have declined due to the introduction of the chytrid fungus Batrachochytrium dendrobatidis (Bd). However, the understanding of the spatiotemporal dynamics remains incomplete. Therefore, estimating the current geographic distribution of Bd is urgently needed, especially in countries like Costa Rica, where susceptible species are still recovering from Bd‐driven declines. We conducted model tuning and spatial analysis to compare the habitat suitability for epizootic and enzootic Bd in Costa Rica and to identify data‐deficient regions, opportunistic sampling, and Bd hotspots. Our dataset combined two methods of detection (histology and PCR methods) for a total of 451 Bd‐positive records from 34 localities. We found that the distribution of enzootic Bd in Costa Rica increased 60% since previous estimates in the early 2000s and extended to highlands and dry lowlands that were considered unsuitable for Bd. We also found that Bd is common across protected lands (80%) and within the herpetological provinces containing the highest amphibian richness and endemism in Costa Rica. Opportunistic sampling of Bd has focused on sites where epizootics occurred with the strongest intensity, leading to deficient or absent sampling across the Talamanca Range, the Nicoya Peninsula, and the northern lowlands. Our results showed that PCR increased the power of Bd detection in lowlands and favored the identification of Bd hotspots across the Caribbean side of Costa Rica. Our results add to the understanding of disease spread during enzootics and can be used to identify new hotspots for disease to mitigate future outbreaks of this pathogen.Abstract in Spanish is available with online material

Occurrence of Batrachochytrium dendrobatidis in Sweden: higher infection prevalence in southern species.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in amphibian populations. While Bd is widespread in southern and central Europe, its occurrence and distribution in northernmost Europe is mostly unknown. We surveyed for Bd in breeding anurans in Sweden by sampling 1917 amphibians from 101 localities and 3 regions in Sweden (southern, northern and central). We found that Bd was widespread in southern and central Sweden, occurring in all 9 investigated species and in 45.5% of the 101 localities with an overall prevalence of 13.8%. No infected individuals were found in the 4 northern sites sampled. The records from central Sweden represent the northernmost records of Bd in Europe. While the proportion of sites positive for Bd was similar between the southern and central regions, prevalence was much higher in the southern region. This was because southern species with a distribution mainly restricted to southernmost Sweden had a higher prevalence than widespread generalist species. The nationally red-listed green toad Bufotes variabilis and the fire-bellied toad Bombina bombina had the highest prevalence (61.4 and 48.9%, respectively). Across species, Bd prevalence was strongly positively, correlated with water temperature at the start of egg laying. However, no individuals showing visual signs of chytridiomycosis were found in the field. These results indicate that Bd is widespread and common in southern and central Sweden with southern species, breeding in higher temperatures and with longer breeding periods, having higher prevalence. However, the impact of Bd on amphibian populations in northernmost Europe remains unknown.

Global Amphibian Declines, Disease, and the Ongoing Battle between Batrachochytrium Fungi and the Immune System

In the approximately 30 years since the recognition of the crisis of global amphibian declines, much has been learned about the likely causes. Among the leading causes are several amphibian diseases including the disease termed chytridiomycosis caused by the chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Here, I briefly review the fundamentals of amphibian immunity, amphibian immune defenses against the chytrid fungi, and the host–pathogen interactions that often favor the pathogen to the detriment of the host. Because amphibians are ectotherms, climate and temperature have a major impact on the amphibian immunity. Thus, I discuss current information about the role that temperature and unpredictable weather events may play in disease and immune responses to the chytrids. Because much research on amphibian declines is directed toward finding management solutions to protect threatened amphibians, I conclude by drawing attention to some of the most promising and novel mitigation strategies that are being proposed.

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA.

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (β=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

Biomarkers of the influence of pyrethroids and neonicotinoids on amphibian larvae

Currently, most amphibian populations in the world exist under the influence of numerous stress factors. Among them, the main factors that affect almost all terrestrial animals, namely, the fragmentation of habitats, environmental pollution and anthropic transformation of landscapes. Moreover, those factors are joined by negative causes that affect only amphibians – specific viral and fungal infections: ranaviruses (Ambystoma tigrinum virus (ATV), Bohle iridovirus (BIV), and frog virus 3) (also dangerous for some reptiles) and chytrid fungi (Batrachochytrium dendrobatidis and B. salamandrivorans). All these factors are one of the main reasons for the current global decline of amphibian populations in the world. In today's world, agricultural chemicals are one of the most important in terms of toxicity, environmental emissions and total area of impact. Among them, a significant proportion are formed by synthetic insecticides, which include pyrethroids and neonicotinoids. Pollution by these substances has a negative impact on amphibian populations, despite the relatively short period of their life in the environment. The vast majority of studies on the effects of pyrethroid and neonicotinoid insecticides were made in laboratory experiments with the larval stages of tailless amphibians. Tadpoles are easy to get in sufficient quantities and kept in the laboratory. Cypermethrin reduces the viability of tadpoles and causes precocious metamorphosis of survived larvae. However, in a combination with other pesticides, it delays metamorphosis. In addition, embryos were more resistant to pyrethroids than tadpoles. Pyrethroid pesticides cause spasms indicating adverse neurological effects. Formation of oral apparatus abnormalities in tadpoles, anisochromasia and increasing number of immature erythrocytes are also caused by pyrethroids. Neonicotinoids show similar effects. Under the action of imidacloprid the erythrocytes are also disturbed that is showed up in the DNA damage and micronuclei formation. Neurological disorders are manifested in the ability to perceive or respond to a predator, disorientation, erratic movement and loss of balance. Pyrethroids and neonicotinoids have been shown cause a variety of disorders: increase mortality and reduce survival of tadpoles; have a teratogenic effect and affect the metamorphosis and morphological parameters of amphibians; change many biochemical parameters that characterize the protein metabolism and oxidative stress; have genotoxic effects and affect the state of the nervous system and animal behaviour. Most of these parameters are proposed for use as biomarkers of pesticide intoxication.

Effects of invasive larval bullfrogs (Rana catesbeiana) on disease transmission, growth and survival in the larvae of native amphibians

The mechanisms by which invasive species negatively affect native species include competition, predation, and the introduction of novel pathogens. Moreover, if an invasive species is a competent disease reservoir, it may facilitate the long-term maintenance and spread of pathogens in ecological assemblages and drive the extinction of less tolerant or less resistant species. Disease-driven loss of biodiversity is exemplified by the amphibian–chytrid fungus system. The disease chytridiomycosis is caused by the aquatic chytrid fungus Batrachochytrium dendrobatidis (Bd) in anurans and is associated with worldwide amphibian population declines and extinctions. For amphibian species that metamorphose and leave infected aquatic habitats, the mechanisms by which Bd persists over winter in these habitats remains a critical open question. A leading hypothesis is that American bullfrogs (Rana catesbeiana), a worldwide invasive species, are tolerant to Bd and serve as a reservoir host for Bd during winter months and subsequently infect native species that return to breed in spring. Using outdoor mesocosms, we experimentally examined if two strains of Bd could overwinter in aquatic systems, in the presence or absence of bullfrog tadpoles, and if overwintered Bd could be transmitted to tadpoles of two spring-breeding species: Pacific treefrogs (Pseudacris regilla) and Cascades frogs (Rana cascadae). We found that only 4 of 448 total animals (one bullfrog and three spring breeders) tested positive for Bd after overwintering. Moreover, two of the three infected spring breeders emerged from tanks that contained overwintered Bd but in the absence of infected bullfrogs. This suggests that Bd can persist over winter without bullfrogs as a reservoir host. We found no effect of Bd strain on bullfrog survival after overwintering. For Pacific treefrogs, Bd exposure did not significantly affect mass at or time to metamorphosis while exposure to bullfrogs reduced survival. For Cascades frogs, we found an interactive effect of Bd strain and bullfrog presence on time to metamorphosis, but no main or interactive effects on their survival or mass at metamorphosis. In short, bullfrog tadpoles rarely retained and transmitted Bd infection in our experiment and we found limited evidence that Bd successfully overwinters in the absence of bullfrog tadpoles and infects spring-breeding amphibians.

Spatiotemporal heterogeneity decouples infection parameters of amphibian chytridiomycosis.

Emerging infectious diseases are responsible for declines in wildlife populations around the globe. Mass mortality events associated with emerging infectious diseases are often associated with high number of infected individuals (prevalence) and high pathogen loads within individuals (intensity). At the landscape scale, spatial and temporal variation in environmental conditions can alter the relationship between these infection parameters and blur the overall picture of disease dynamics. Quantitative estimates of how infection parameters covary with environmental heterogeneity at the landscape scale are scarce. If we are to identify wild populations at risk of disease epidemics, we must elucidate the factorsthat shape, and potentially decouple, the link between pathogen prevalence and intensity of infection over complex ecological landscapes. Using a network of 41 populations of the amphibian host Rana pipiens in Ontario, Canada, we present the spatial and temporal heterogeneity in pathogen prevalence and intensity of infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd), across a 3-year period. We then quantify how covariation between both infection parameters measured during late summer is modified by previously experienced spatiotemporal environmental heterogeneity across 14 repeat sampled populations. Late summer Bd infection parameters are governed, at least in part, by different environmental factors operating during separate host life-history events. Our results provide evidence for a relationship between Bd prevalence and thermal regimes prior to host breeding at the site level, and a relationship between intensity of infection and aquatic conditions (precipitation, hydroshed size and river density) throughout host breeding period at the site level. This demonstrates that microclimatic variation within temporal windows can drive divergent patterns of pathogen dynamics within and across years, by effecting changes in host behaviour which interfere with the pathogen's ability to infect and re-infect hosts. A clearer understanding of the role that spatiotemporal heterogeneity has upon infection parameters will provide valuable insights into host-pathogen epidemiology, as well as more fundamental aspects of the ecology and evolution of interspecific interactions.

Discovery of granulocyte-lineage cells in the skin of the amphibianXenopus laevis

The ranavirus Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis ( Bd) are significant contributors to the global amphibian declines and both pathogens target the amphibian skin. We previously showed that tadpoles and adults of the anuran amphibian Xenopus laevis express notable levels of granulocyte chemokine genes ( cxcl8a and cxcl8b) within their skin and likely possess skin-resident granulocytes. Presently, we show that tadpole and adult X. laevis indeed possess granulocyte-lineage cells within their epidermises that are distinct from their skin mast cells, which are found predominantly in lower dermal layers. These esterase-positive cells responded to (r)CXCL8a and rCXCL8b in a concentration- and CXCR1/CXCR2-dependent manner, possessed polymorphonuclear granulocyte morphology, granulocyte marker surface staining, and exhibited distinct immune gene expression from conventional granulocytes. Our past work indicates that CXCL8b recruits immunosuppressive granulocytes, and here we demonstrated that enriching esterase-positive skin granulocytes with rCXCL8b (but not rCXCL8a) may increase tadpole susceptibility to FV3 and adult frog susceptibility to Bd. Furthermore, pharmacological depletion of skin-resident granulocytes increased tadpole susceptibility to FV3. This manuscript provides new insights into the composition and roles of immune cells within the amphibian skin, which is a critical barrier against pathogenic contributors to the amphibian declines.

Effects of Snowpack, Temperature, and Disease on Demography in a Wild Population of Amphibians

Understanding the demographic consequences of interactions among pathogens, hosts, and weather conditions is critical in determining how amphibian populations respond to disease and in identifying site-specific conservation actions that can be developed to bolster persistence of amphibian populations. We investigated population dynamics in Boreal Toads (Anaxyrus boreas) relative to abiotic (fall temperatures and snowpack) and biotic (the abundance of another anuran host and disease) characteristics of the local environment in Wyoming, USA. We used capture–recapture data and a multistate model where state was treated as a hidden Markov process to incorporate disease state uncertainty and assess our a priori hypotheses. Our results indicated that snowpack during the coldest week of winter is more influential to toad survival, disease transition probabilities, and the population-level prevalence of the amphibian chytrid fungus (Batrachochytrium dendrobatidis) in the spring, than temperatures in the fall or the presence of another host. As hypothesized, apparent survival at low (i.e., <25 cm) snowpack (0.22; confidence interval [CI] = 0.15–0.31) was lower than apparent survival at high snowpack (90.65; CI = 0.50–0.78). Our findings highlight the potential for local environmental factors, like snowpack, to influence disease and host persistence, and demonstrate the ecological complexity of disease effects on population demography in natural environments. This work further emphasizes the need for improved understanding of how climate change may influence the relationships among pathogens, hosts, and their environment for wild animal populations challenged by disease.

Reference gene screening of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans for quantitative real-time PCR studies

Real-time quantitative PCR studies largely depend on reference genes for the normalization of gene expression. Stable reference genes should be accurately selected in order to obtain reliable results. We here present a study screening commonly used reference genes (TEF1F, α-centractin, Ctsyn1, GAPDH, R6046, APRT and TUB) in the chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), which cause the lethal amphibian skin disease chytridiomycosis. We evaluated the stability of the reference gene candidates during different growth stages of the fungi, using different statistical software packages: ΔCT, BestKeeper, GeNorm, NormFinder and RefFinder. In order to reflect the in vivo situation, the stability of the candidates was assessed when taking all growth stages into account. Using an ex-vivo approach, we tested whether the expression of GAPDH, TUB, R6046 and APRT (Bd) and GAPDH, TUB, R6046 and α-centractin (Bsal) remained stable when these fungi came in contact with host tissue. Finally, their role as in vivo reference genes was examined in skin tissue of experimentally infected midwife toads (Alytes obstetricans) (Bd) and fire salamanders (Salamandra salamandra) (Bsal). Summarized, the present study provides guidance for selecting appropriate reference genes when analyzing expression patterns of these fungal organisms during different growth stages and in Bd- or Bsal-infected tissues.

Fungused frogs fail to fix fast fluid flux

![Graphic][1] Amphibian populations are facing a crisis – the chytrid fungus Batrachochytrium dendrobatidis is responsible for the decline of more than 500 species around the world. This fungal infection targets the skin of amphibians, making it leaky and susceptible to water loss. To fight

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Amphibian chytrid fungus in Africa – realigning hypotheses and the research paradigm

AbstractThe amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), responsible for numerous amphibian declines and extinctions, was previously thought to originate from the African continent. This was based on infected museum specimens from early 20th century South Africa, Cameroon and Uganda. Further research on archived specimens from other continents eventually revealed early 20th century records also in Brazil and Japan. Recent robust analysis of genomic diversity and phylogeny of Bd has shown origin from Asia to be more plausible. This raises the issue that the threat of Bd for African amphibians as a novel pathogen has been underestimated. There are now cases where dramatic amphibian declines in disparate mountains on the continent could be attributed to Bd, and this necessitates an urgent realigning of hypotheses and the research agenda for amphibian conservation on the continent. Notably, hotspots of amphibian host naivety include West Africa where this pathogen has so far not been detected. We discuss research gaps that amphibian conservationists might focus on, notably more genomic sequencing of the BdCAPE (the less virulent) lineage to determine its date of emergence, and assessing the susceptibility of different amphibian species to infection, disease and decline to better prioritize conservation actions.