Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions.

Ancient enzootic associations between wildlife and their infections allow evolution to innovate mechanisms of pathogenicity that are counterbalanced by host responses. However, erosion of barriers to pathogen dispersal by globalization leads to the infection of hosts that have not evolved effective resistance and the emergence of highly virulent infections. Global amphibian declines driven by the rise of chytrid fungi and chytridiomycosis are emblematic of emerging infections. Here, we review how modern biological methods have been used to understand the adaptations and counteradaptations that these fungi and their amphibian hosts have evolved. We explore the interplay of biotic and abiotic factors that modify the virulence of these infections and dissect the complexity of this disease system. We highlight progress that has led to insights into how we might in the future lessen the impact of these emerging infections.

Rapid Evolution of Resistance and Tolerance Leads to Variable Host Recoveries following Disease-Induced Declines.

AbstractRecoveries of populations that have suffered severe disease-induced declines are being observed across disparate taxa. Yet we lack theoretical understanding of the drivers and dynamics of recovery in host populations and communities impacted by infectious disease. Motivated by disease-induced declines and nascent recoveries in amphibians, we developed a model to ask the following question: How does the rapid evolution of different host defense strategies affect the transient recovery trajectories of hosts following pathogen invasion and disease-induced declines? We found that while host life history is predictably a major driver of variability in population recovery trajectories (including declines and recoveries), populations that use different host defense strategies (i.e., tolerance, avoidance resistance, and intensity-reduction resistance) experience notably different recoveries. In single-species host populations, populations evolving tolerance recovered on average four times slower than populations evolving resistance. Moreover, while populations using avoidance resistance strategies had the fastest potential recovery rates, these populations could get trapped in long transient states at low abundance prior to recovery. In contrast, the recovery of populations evolving intensity-reduction resistance strategies were more consistent across ecological contexts. Overall, host defense strategies strongly affect the transient dynamics of population recovery and may affect the ultimate fate of real populations recovering from disease-induced declines.

Centrifugation is an effective and inexpensive way to determine Batrachochytrium dendrobatidis quantity in water samples with low turbidity.

Batrachochytrium dendrobatidis (Bd) is a pathogenic chytrid fungus that is particularly lethal for amphibians. Bd can extirpate amphibian populations within a few weeks and remain in water in the absence of amphibian hosts. Most efforts to determine Bd presence and quantity in the field have focused on sampling hosts, but these data do not give us a direct reflection of the amount of Bd in the water, which are useful for parameterizing disease models, and are not effective when hosts are absent or difficult to sample. Current methods for screening Bd presence and quantity in water are time, resource, and money intensive. Here, we developed a streamlined method for detecting Bd in water with low turbidity (e.g., water samples from laboratory experiments and relatively clear pond water from a natural lentic system). We centrifuged water samples with known amounts of Bd to form a pellet and extracted the DNA from that pellet. This method was highly effective and the resulting concentrations across all tested treatments presented a highly linear relationship with the expected values. While the experimentally derived values were lower than the inoculation doses, the values were highly correlated and a conversion factor allows us to extrapolate the actual Bd concentration. This centrifuge-based method is effective, repeatable, and would greatly expand the domain of tractable questions to be explored in the field of Bd ecology. Importantly, this method increases equity in the field, because it is time- and cost-efficient and requires few resources.

Evidence for a metal disease refuge: The amphibian-killing fungus (Batrachochytrium dendrobatidis) is inhibited by environmentally-relevant concentrations of metals tolerated by amphibians.

The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has caused substantial declines in Bd-susceptible amphibian species worldwide. However, some populations of Bd-susceptible frogs have managed to survive at existing metal-polluted sites, giving rise to the hypothesis that frogs might persist in the presence of Bd if Bd is inhibited by metals at concentrations that frogs can tolerate. We tested this hypothesis by measuring the survival of Bd zoospores, the life stage that infects amphibians, and calculated the LC50 after exposure to environmentally-relevant elevated concentrations of copper (Cu), zinc (Zn), and their combination (Cu + Zn) in two repeated 4-day acute exposure runs. We also measured the chronic sensitivity of Bd to these metals over three generations by measuring the number of colonies and live zoospores and calculating EC50 concentrations after 42 days of exposure. We then compared acute and chronic sensitivity of Bd with amphibian sensitivities by constructing species sensitivity distributions (SSDs) using LC50 and EC50 data obtained from the literature. Acute sensitivity data showed that Bd zoospore survival decreased with increasing metal concentrations and exposure durations relative to the control, with the highest LC50 values for Cu and Zn being 2.5 µg/L and 250 µg/L, respectively. Chronic exposures to metals resulted in decreased numbers of Bd colonies and live zoospores after 42 days, with EC50 values of 0.75 µg/L and 1.19 µg/L for Cu and Zn, respectively. Bd zoospore survival was 10 and 8 times more sensitive to Cu and Zn, respectively in acute, and 2 and 5 times more sensitive to Cu and Zn in chronic exposure experiments than the most sensitive amphibian species recorded. Our findings are consistent with the hypothesis that metals in existing metal-polluted sites may have a greater impact on Bd relative to amphibians' performance, potentially enabling Bd-susceptible amphibians to persist with Bd at these sites.

Phytoplankton-chytrid-zooplankton dynamics via a reaction-diffusion-advection mycoloop model.

Mycoloop is an important aquatic food web composed of phytoplankton, chytrids (one dominant group of parasites in aquatic ecosystems), and zooplankton. Chytrids infect phytoplankton and fragment them for easy consumption by zooplankton. The free-living chytrid zoospores are also a food resource for zooplankton. A dynamic reaction-diffusion-advection mycoloop model is proposed to describe the Phytoplankton-chytrid-zooplankton interactions in a poorly mixed aquatic environment. We analyze the dynamics of the mycoloop model to obtain dissipativity, steady state solutions, and persistence. We rigorously derive several critical thresholds for phytoplankton or zooplankton invasion and chytrid transmission among phytoplankton. Numerical diagrams show that varying ecological factors affect the formation and breakup of the mycoloop, and zooplankton can inhibit chytrid transmission among phytoplankton. Furthermore, this study suggests that mycoloop may either control or cause phytoplankton blooms.

Countrywide screening supports model-based predictions of the distribution of Batrachochytrium dendrobatidis in Ukraine.

The chytrid Batrachochytrium dendrobatidis (Bd) is a widespread fungus causing amphibian declines across the globe. Although data on Bd occurrence in Eastern Europe are scarce, a recent species distribution model (SDM) for Bd reported that western and north-western parts of Ukraine are highly suitable to the pathogen. We verified the SDM-predicted range of Bd in Ukraine by sampling amphibians across the country and screening for Bd using qPCR. A total of 446 amphibian samples (tissue and skin swabs) from 11 species were collected from 36 localities. We obtained qPCR-positive results for 33 samples including waterfrogs (Pelophylax esculentus complex) and fire- and yellow-bellied toads (Bombina spp.) from 8 localities. We found that Bd-positive localities had significantly higher predicted Bd habitat suitability than sites that were pathogen-free. Amplification and sequencing of the internal transcribed spacer (ITS) region of samples with the highest Bd load revealed matches with ITS haplotypes of the globally distributed BdGPL strain, and a single case of the BdASIA-2/BdBRAZIL haplotype. We found that Bd was non-randomly distributed across Ukraine, with infections present in the western and north-central forested peripheries of the country with a relatively cool, moist climate. On the other hand, our results suggest that Bd is absent or present in low abundance in the more continental central, southern and eastern regions of Ukraine, corroborating the model-predicted distribution of chytrid fungus. These areas could potentially serve as climatic refugia for Bd-susceptible amphibian hosts.

Widespread occurrence of the amphibian chytrid panzootic lineage in Uruguay is constrained by climate.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease among the main causes of amphibian declines worldwide. However, Bd studies on Neotropical amphibians from temperate areas are scarce. We present a comprehensive survey of Bd in Uruguay, in temperate central eastern South America, carried out between 2006 and 2014. Skin swabs of 535 specimens of 21 native and exotic frogs were tested by PCR. We used individual-level data to examine the relationship between infection, climatic variables, and their effects on body condition and the number of prey items found in stomach contents. Infection was widespread in free-ranging anurans with an overall prevalence of 41.9%, detected in 15 native species, wild American bullfrogs Aquarana catesbeiana, and captive specimens of Ceratophrys ornata and Xenopus laevis. Three haplotypes of the Bd ITS region were identified in native amphibians, all belonging to the global panzootic lineage (BdGPL), of which only one was present in exotic hosts. Despite high infection frequencies in different anurans, we found no evidence of morbidity or mortality attributable to chytridiomycosis, and we observed no discernible impact on body condition or consumed prey. Climatic conditions at the time of our surveys suggested that the chance of infection is associated with monthly mean temperature, mean humidity, and total precipitation. Temperatures below 21°C combined with moderate humidity and pronounced rainfall may increase the likelihood of infection. Multiple haplotypes of BdGPL combined with high frequencies of infection suggest an enzootic pattern in native species, underscoring the need for continued monitoring.

Glove decontamination procedures to prevent pathogen and DNA cross-contamination among frogs.

Working with aquatic organisms often requires handling multiple individuals in a single session, potentially resulting in cross-contamination by live pathogens or DNA. Most researchers address this problem by disposing of gloves between animals. However, this generates excessive waste and may be impractical for processing very slippery animals that might be easier to handle with cotton gloves. We tested methods to decontaminate cotton or nitrile gloves after contamination with cultured Batrachochytrium dendrobatidis (Bd) or after handling heavily Bd-infected Xenopus laevis with layered cotton and nitrile gloves. Bleach eliminated detectable Bd DNA from culture-contaminated nitrile gloves, but gloves retained detectable Bd DNA following ethanol disinfection. After handling a Bd-infected frog, Bd DNA contamination was greatly reduced by removal of the outer cotton glove, after which either bleach decontamination or ethanol decontamination followed by drying hands with a paper towel lowered Bd DNA below the detection threshold of our assay. These results provide new options to prevent pathogen or DNA cross-contamination, especially when handling slippery aquatic organisms. However, tradeoffs should be considered when selecting an animal handling procedure, such as the potential for cotton gloves to abrade amphibian skin or disrupt skin mucus. Disposing of gloves between animals should remain the gold standard for maintaining biosecurity in sensitive situations.

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.

Ontogeny drives shifts in skin bacterial communities in facultatively paedomorphic salamanders.

Microbiomes are major determinants of host growth, development and survival. In amphibians, host-associated bacteria in the skin can inhibit pathogen infection, but many processes can influence the structure and composition of the community. Here we quantified the shifts in skin-associated bacteria across developmental stages in the striped newt (Notophthalmus perstriatus), a threatened salamander species with a complex life history and vulnerable to infection by the amphibian chytrid fungus Batrachochytrium dendrobatidis and ranavirus. Our analyses show that pre-metamorphic larval and paedomorphic stages share similar bacterial compositions, and that the changes in the microbiome coincided with physiological restructuring during metamorphosis. Newts undergoing metamorphosis exhibited microbiome compositions that were intermediate between paedomorphic and post-metamorphic stages, further supporting the idea that metamorphosis is a major driver of host-associated microbes in amphibians. We did not find support for infection-related disruption of the microbiome, though infection replicates were small for each respective life stage.

Empirical evidence for effects of invasive American Bullfrogs on occurrence of native amphibians and emerging pathogens.

Invasive species and emerging infectious diseases are two of the greatest threats to biodiversity. American Bullfrogs (Rana [Lithobates] catesbeiana), which have been introduced to many parts of the world, are often linked with declines in native amphibians via predation and the spread of emerging pathogens such as amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]) and ranaviruses. Although many studies have investigated the potential role of bullfrogs in the decline of native amphibians, analyses that account for shared habitat affinities and imperfect detection have found limited support for clear effects. Similarly, the role of bullfrogs in shaping the patch-level distribution of pathogens is unclear. We used eDNA methods to sample 233 sites in the southwestern USA and Sonora, Mexico (2016-2018) to estimate how the presence of bullfrogs affects the occurrence of four native amphibians, Bd, and ranaviruses. Based on two-species, dominant-subordinate occupancy models fitted in a Bayesian context, federally threatened Chiricahua Leopard Frogs (Rana chiricahuensis) and Western Tiger Salamanders (Ambystoma mavortium) were eight times (32% vs. 4%) and two times (36% vs. 18%), respectively, less likely to occur at sites where bullfrogs occurred. Evidence for the negative effects of bullfrogs on Lowland Leopard Frogs (Rana yavapaiensis) and Northern Leopard Frogs (Rana pipiens) was less clear, possibly because of smaller numbers of sites where these native species still occurred and because bullfrogs often occur at lower densities in streams, the primary habitat for Lowland Leopard Frogs. At the community level, Bd was most likely to occur where bullfrogs co-occurred with native amphibians, which could increase the risk to native species. Ranaviruses were estimated to occur at 33% of bullfrog-only sites, 10% of sites where bullfrogs and native amphibians co-occurred, and only 3% of sites where only native amphibians occurred. Of the 85 sites where we did not detect any of the five target amphibian species, we also did not detect Bd or ranaviruses; this suggests other hosts do not drive the distribution of these pathogens in our study area. Our results provide landscape-scale evidence that bullfrogs reduce the occurrence of native amphibians and increase the occurrence of pathogens, information that can clarify risks and aid the prioritization of conservation actions.

Pseudacris regilla metamorphs acquire resistance to Batrachochytrium dendrobatidis after exposure to the killed fungus.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is associated with drastic global amphibian declines. Prophylactic exposure to killed zoospores and the soluble chemicals they produce (Bd metabolites) can induce acquired resistance to Bd in adult Cuban treefrogs Osteopilus septentrionalis. Here, we exposed metamorphic frogs of a second species, the Pacific chorus frog Pseudacris regilla, to one of 2 prophylactic treatments prior to live Bd exposures: killed Bd zoospores with metabolites, killed zoospores alone, or a water control. Prior exposure to killed Bd zoospores with metabolites reduced Bd infection intensity in metamorphic Pacific chorus frogs by 60.4% compared to control frogs. Interestingly, Bd intensity in metamorphs previously exposed to killed zoospores alone did not differ in magnitude relative to the control metamorphs, nor to those treated with killed zoospores plus metabolites. Previous work indicated that Bd metabolites alone can induce acquired resistance in tadpoles, and so these findings together indicate that it is possible that the soluble Bd metabolites may contain immunomodulatory components that drive this resistance phenotype. Our results expand the generality of this prophylaxis work by identifying a second amphibian species (Pacific chorus frog) and an additional amphibian life stage (metamorphic frog) that can acquire resistance to Bd after metabolite exposure. This work increases hopes that a Bd-metabolite prophylaxis might be widely effective across amphibian species and life stages.

Effects of Herbicides and the Chytrid Fungus Batrachochytrium dendrobatidis on the growth, development and survival of Larval American Toads (Anaxyrus americanus).

Pesticides and pathogens adversely affect amphibian health, but their interactive effects are not well known. We assessed independent and combined effects of two agricultural herbicides and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on the growth, development and survival of larval American toads (Anaxyrus americanus). Wild-caught tadpoles were exposed to four concentrations of atrazine (0.18, 1.8, 18.0, 180 µg/L) or glyphosate (7, 70, 700, 7000 µg a.e./L), respectively contained in Aatrex® Liquid 480 (Syngenta) or Vision® Silviculture Herbicide (Monsanto) for 14 days, followed by two doses of Bd. At day 14, atrazine had not affected survival, but it non-monotonically affected growth. Exposure to the highest concentration of glyphosate caused 100% mortality within 4 days, while lower doses had an increasing monotonic effect on growth. At day 65, tadpole survival was unaffected by atrazine and the lower doses of glyphosate. Neither herbicide demonstrated an interaction effect with Bd on survival, but exposure to Bd increased survival among both herbicide-exposed and herbicide-control tadpoles. At day 60, tadpoles exposed to the highest concentration of atrazine remained smaller than controls, indicating longer-term effects of atrazine on growth, but effects of glyphosate on growth disappeared. Growth was unaffected by any herbicide-fungal interaction but was positively affected by exposure to Bd following exposure to atrazine. Atrazine exhibited a slowing and non-monotonic effect on Gosner developmental stage, while exposure to Bd tended to speed up development and act antagonistically toward the observed effect of atrazine. Overall, atrazine, glyphosate and Bd all showed a potential to modulate larval toad growth and development.

First Report of False Rust caused by Synchytrium psophocarpi on Winged Bean (Psophocarpus tetragonolobus) in Taiwan.

From September 2020 to January 2021, an unknown disease of winged bean (Psophocarpus tetragonolobus) was reported by local growers in the Toucheng Town, Yilan County (N24.91, E121.85). The disease occurs in all age of winged bean, and the occurrence tended to be higher in humid environment, such as branches in lower canopy or branches in high density. The disease symptoms, which also appeared to be the sign of the pathogen, were spherical pustules in yellow to orange color on the stems, leaves, and pods of winged bean. Severely infected plants also exhibited growth reduction, malformation, and curling of the leaves and pods. According to the disease literature of winged bean, this unknown disease was likely to be the false rust caused by a chytrid pathogen, Synchytrium psophocarpi (UK, CAB International. 1993); and the uredinia-liked pustules could be the sori, which contain numerous ovoid to globose sporangia inside. In order to characterize the pathogen identity, the sori were manually ruptured to assess the size of individual sporangium, which had an average of 26.71 ± 4.25 µm x 26.61 ± 4.60 µm (n=42), similar to the size reported in literature (Drinkall and Price. 1979). To confirm the molecular identity, the full genomic sequences from the small subunit (SSU) to the internal transcribed spacer-1 (ITS-1), 5.8S unit, and ITS-2 were amplified using the primer sets NS3 and ITS4. The 2,263 bp amplicon was cloned and sequenced to reveal the identity (Smith et al. 2014). The BLASTN results matched the SSU of our isolate (MW649126.1) to the Synchytrium minutum (HQ324138.1) with 96% similarity (1,075 out of 1,121 bp in length), Synchytrium decipiens isolate DAOM_87618 (KF160868.1) with 92% similarity (1,215 out of 1,326 bp in length) and S. decipiens isolate AFTOL-ID 634 (DQ536475.1) with 92% similarity (1210 out of 1316 bp in length). Phylogenetic analysis using the SSU sequence revealed this unknown pathogen was the grouped within the clade of Synchytrium genus with 100% bootstrapping confidence (Smith et al. 2014). Accordingly, the pathogen was confirmed to be a Synchytrium chytrid fungus. To complete the Koch's postulates, the sori were collected from infected tissue. After vortexing washing in 1% bleach for surface sterilization, the sori were gently crashed by a plastic tube pestle to harvest sporangia. The sporangia were sprayed onto healthy winged beans cultivated in pots, and the inoculated plants were kept in a moisture bag in 25 °C. While leaf curling and malformation could be observed about 14 days post inoculation, the yellow to orange sori could be observed around 30 to 40 days post inoculation on the whole plants cultivated in pots. The sori were collected to confirm the sporangia and the sequences were identical to the original pathogen. Collectively, this study not only presents the first report for the false rust of winged bean in Taiwan, but also documents the first reference sequence of S. psophocarpi that will be useful for future molecular diagnosis. Since S. psophocarpi has been only reported in tropic regions including Indonesia, Malay Peninsula, Malaysia, Papua New Guinea, and Philippines, this report provides the first observation of S. psophocarpi moving in the subtropic region.

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

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

Inhibitory Bacterial Diversity and Mucosome Function Differentiate Susceptibility of Appalachian Salamanders to Chytrid Fungal Infection.

Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), in four salamander species with different Batrachochytrium susceptibilities. Bd infection was quantified using qPCR, mucosome function (i.e., ability to kill Bd or Bsal zoospores in vitro), skin bacterial communities using 16S rRNA gene amplicon sequencing, and the role of Bd-inhibitory bacteria in microbial networks across all species. We explored the presence of candidate-AMPs in eastern newts and red-backed salamanders. Eastern newts had the highest Bd prevalence and mucosome function, while red-back salamanders had the lowest Bd prevalence and mucosome function, and two-lined salamanders and seal salamanders were intermediates. Salamanders with highest Bd infection intensity showed greater mucosome function. Bd infection prevalence significantly decreased as putative Bd-inhibitory bacterial richness and relative abundance increased on hosts. In co-occurrence networks, some putative Bd-inhibitory bacteria were found as hub-taxa, with red-backs having the highest proportion of protective hubs and positive associations related to putative Bd-inhibitory hub bacteria. We found more AMP candidates on salamanders with lower Bd susceptibility. These findings suggest that salamanders possess distinct innate mechanisms that affect chytrid fungi. IMPORTANCE How host mucosal defenses interact, and influence disease outcome is critical in understanding host defenses against pathogens. A more detailed understanding is needed of the interactions between the host and the functioning of its mucosal defenses in pathogen defense. This study investigates the variability of chytrid susceptibility in salamanders and the innate defenses each species possesses to mediate pathogens, thus advancing the knowledge toward a deeper understanding of the microbial ecology of skin-associated bacteria and contributing to the development of bioaugmentation strategies to mediate pathogen infection and disease. This study improves the understanding of complex immune defense mechanisms in salamanders and highlights the potential role of the mucosome to reduce the probability of Bd disease development and that putative protective bacteria may reduce likelihood of Bd infecting skin.

Taxonomic and functional dynamics during chytrid epidemics in an aquatic ecosystem.

Fungal parasitism is common in plankton communities and plays a crucial role in the ecosystem by balancing nutrient cycling in the food web. Previous studies of aquatic ecosystems revealed that zoosporic chytrid epidemics represent an important driving factor in phytoplankton seasonal successions. In this study, host-parasite dynamics in Lake Pavin (France) were investigated during the spring diatom bloom while following chytrid epidemics using next generation sequencing (NGS). Metabarcoding analyses were applied to study changes in the eukaryotic microbial community throughout diatom bloom-chytrid epidemics. Relative read abundances of metabarcoding data revealed potential "beneficiaries" and "victims" during the studied period. Subsequently, metatranscriptomic analyses on samples before and during the chytrid epidemic unveiled the active part of the community and functional/metabolic dynamics in association with the progress of chytrid infection. Diatom functions involving lipases, transporters, histones, vacuolar systems, the proteasome, proteases and DNA/RNA polymerases were more abundant during the diatom bloom. Chytrid functions related to a parasitic lifestyle including invasion, colonization and stress tolerance were up-regulated during the chytrid epidemic. In addition, functions related to the degradation/metabolism of proteins, lipids and chitin were in higher proportion in the community during the epidemic event. Results of NGS and bioinformatics analyses offered a panorama of dynamic biodiversity and biological functioning of the community.

Parasites do not adapt to elevated temperature, as evidenced from experimental evolution of a phytoplankton-fungus system.

Global warming is predicted to impact the prevalence and severity of infectious diseases. However, empirical data supporting this statement usually stem from experiments in which parasite fitness and disease outcome are measured directly after temperature increase. This might exclude the possibility of parasite adaptation. To incorporate the adaptive response of parasites into predictions of disease severity in a warmer world, we undertook an experimental evolution assay in which a fungal parasite of phytoplankton was maintained at elevated or control temperatures for six months, corresponding to 100-200 parasite generations. Host cultures were maintained at the respective temperatures and provided as substrate, but were not under parasite pressure. A reciprocal infection experiment conducted after six-month serial passages revealed no evidence of parasite adaptation. In fact, parasite fitness at elevated temperatures was inferior in parasite populations reared at elevated temperatures compared with those maintained under control temperature. However, this effect was reversed after parasites were returned to control temperatures for a few (approx. 10) generations. The absence of parasite adaptation to elevated temperatures suggests that, in phytoplankton-fungus systems, disease outcome under global warming will be largely determined by both host and parasite thermal ecology.

Infectious disease threats to amphibians in Greece: new localities positive for Batrachochytrium dendrobatidis.

In the early 2000s, numerous cases of European amphibian population declines and mass die-offs started to emerge. Investigating those events led to the discovery that wild European amphibians were confronted with grave disease threats caused by introduced pathogens, namely the amphibian and the salamander chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) and ranaviruses. In Greece, Bd was previously documented among wild amphibian populations in 2 different locations and 3 different species. However, no disease-related mass declines or mortality events have been reported. In this work, we build upon previous findings with new, subsequently obtained data, resulting in a 225-sample dataset of 14 species from 17 different locations throughout Greece, in order to examine the occurrence status of all 3 pathogens responsible for emerging infectious diseases in European amphibians. No positive samples for Bsal or ranavirus were recorded in any location. We confirmed the presence of Bd in 4 more localities and in 4 more species, including 1 urodelan (Macedonian crested newt Triturus macedonicus) and 1 introduced anuran (American bullfrog Lithobates catesbeianus). All insular localities were negative for Bd, except for Crete, where Bd was identified in 2 different locations. Again, no mass declines or die-offs were recorded in any Bd-positive area or elsewhere. However, given the persistence of Bd across Greece over the past ~20 yr, monitoring efforts should continue, and ideally be further expanded.