Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Preparatory immunity: Seasonality of mucosal skin defences and Batrachochytrium infections in Southern leopard frogs.

Accurately predicting the impacts of climate change on wildlife health requires a deeper understanding of seasonal rhythms in host-pathogen interactions. The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), exhibits seasonality in incidence; however, the role that biological rhythms in host defences play in defining this pattern remains largely unknown. The aim of this study was to examine whether host immune and microbiome defences against Bd correspond with infection risk and seasonal fluctuations in temperature and humidity. Over the course of a year, five populations of Southern leopard frogs (Rana [Lithobates] sphenocephala) in Tennessee, United States, were surveyed for host immunity, microbiome and pathogen dynamics. Frogs were swabbed for pathogen load and skin bacterial diversity and stimulated to release stored antimicrobial peptides (AMPs). Secretions were analysed to estimate total hydrophobic peptide concentrations, presence of known AMPs and effectiveness of Bd growth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibit Bd growth in vitro were used as an estimate of predicted anti-Bd function of the skin microbiome. Batrachochytrium dendrobatidis dynamics followed the expected seasonal fluctuations-peaks in cooler months-which coincided with when host mucosal defences were most potent against Bd. Specifically, the concentration and expression of stored AMPs cycled synchronously with Bd dynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibit Bd growth was greatest when risk of Bd infection was highest. We interpret the increase in peptide storage in the fall and the shift to a more anti-Bd microbiome over winter as a preparatory response for subsequent infection risk during the colder periods when AMP synthesis and bacterial growth is slow and pathogen pressure from this cool-adapted fungus is high. Given that a decrease in stored AMP concentrations as temperatures warm in spring likely means greater secretion rates, the subsequent decrease in prevalence suggests seasonality of Bd in this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.

Extraction-free detection of amphibian pathogens from water baths.

Detecting and quantifying pathogens with quick, cost-efficient and sensitive methods is needed across disease systems for addressing pertinent epidemiological questions. Typical methods rely on extracting DNA from collected samples. Here we develop and test an extraction-free method from water bath samples that is both sensitive and efficient for 2 major amphibian pathogens-Batrachochytrium dendrobatidis and B. salamandrivorans. We tested mock samples with known pathogen quantities as well as comparatively assessed detection from skin swabs and water baths from field sampled amphibians. Quantitative PCR (qPCR) directly on lyophilized water baths was able to reliably detect low loads of 10 and 1 zoospores for both pathogens, and detection rates were greater than those of swabs from field samples. Further concentration of samples did not improve detection, and collection container type did not influence pathogen load estimates. This method of lyophilization (i.e. freeze-drying) followed by direct qPCR offers an effective and efficient tool from detecting amphibian pathogens, which is crucial for surveillance efforts and estimating shedding rates for robust epidemiological understanding of transmission dynamics. Furthermore, water bath samples have multiple functions and can be used to evaluate mucosal function against pathogens and characterize mucosal components. The multifunctionality of water bath samples and reduced monetary costs and time expenditures make this method an optimal tool for amphibian disease research and may also prove to be useful in other wildlife disease systems.

Responsible biosecurity and risk mitigation for laboratory research on emerging pathogens of amphibians.

The increasing study of emerging wildlife pathogens and a lack of policy or legislation regulating their translocation and use has heightened concerns about laboratory escape, species spillover, and subsequent epizootics among animal populations. Responsible self-regulation by research laboratories, in conjunction with institutional-level safeguards, has an important role in mitigating pathogen transmission and spillover, as well as potential interspecies pathogenesis. A model system in disease ecology that highlights these concerns and related amelioration efforts is research focused on amphibian emerging infectious diseases. Whereas laboratory escape of amphibian pathogens has not been reported and may be rare compared with introduction events from trade or human globalization, the threat that novel disease outbreaks with mass mortality effects pose to wild populations warrants thorough biosecurity measures to ensure containment and prevent spillover. Here, we present a case study of the laboratory biosecurity concerns for the emerging amphibian fungal pathogen Batrachochytrium salamandrivorans. We conclude that proactive biosecurity strategies are needed to integrate researcher and institutional oversight of aquatic wildlife pathogens generally, and we call for increased national and international policy and legislative enforcement. Furthermore, taking professional responsibility beyond current regulations is needed as development of legal guidance can be slow at national and international levels. We outline the need for annual laboratory risk assessments, comprehensive training for all laboratory personnel, and appropriate safeguards specific to pathogens under study. These strategies are critical for upholding the integrity and credibility of the scientific community and maintaining public support for research on wildlife diseases.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

Prevalence and pathogen load estimates for the fungus Batrachochytrium dendrobatidis are impacted by ITS DNA copy number variation.

The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibians and white nose syndrome in bats. Chytridiomycosis is caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and is responsible for declines and extinctions of amphibians worldwide. Over a decade ago, a qPCR assay was developed to determine Bd prevalence and pathogen load. Here, we demonstrate the effect that ITS copy number variation in Bd strains can have on the estimation of prevalence and pathogen load. We used data sets from different amphibian species to simulate how ITS copy number affects prevalence and pathogen load. In addition, we tested 2 methods (gBlocks® synthetic standards and digital PCR) to determine ITS copy number in Bd strains. Our results show that assumptions about the ITS copy number can lead to under- or overestimation of Bd prevalence and pathogen load. The use of synthetic standards replicated previously published estimates of ITS copy number, whereas dPCR resulted in estimates that were consistently lower than previously published estimates. Standardizing methods will assist with comparison across studies and produce reliable estimates of prevalence and pathogen load in the wild, while using the same Bd strain for exposure experiments and zoospore standards in qPCR remains the best method for estimating parameters used in epidemiological studies.

Shotgun metagenomics captures more microbial diversity than targeted 16S rRNA gene sequencing for field specimens and preserved museum specimens.

The use of museum specimens for research in microbial evolutionary ecology remains an under-utilized investigative dimension with important potential. Despite this potential, there remain barriers in methodology and analysis to the wide-spread adoption of museum specimens for such studies. Here, we hypothesized that there would be significant differences in taxonomic prediction and related diversity among sample type (museum or fresh) and sequencing strategy (medium-depth shotgun metagenomic or 16S rRNA gene). We found dramatically higher predicted diversity from shotgun metagenomics when compared to 16S rRNA gene sequencing in museum and fresh samples, with this differential being larger in museum specimens. Broadly confirming these hypotheses, the highest diversity found in fresh samples was with shotgun sequencing using the Rep200 reference inclusive of viruses and microeukaryotes, followed by the WoL reference database. In museum-specimens, community diversity metrics also differed significantly between sequencing strategies, with the alpha-diversity ACE differential being significantly greater than the same comparisons made for fresh specimens. Beta diversity results were more variable, with significance dependent on reference databases used. Taken together, these findings demonstrate important differences in diversity results and prompt important considerations for future experiments and downstream analyses aiming to incorporate microbiome datasets from museum specimens.

Parental care contributes to vertical transmission of microbes in a skin-feeding and direct-developing caecilian.

BACKGROUND: Our current understanding of vertebrate skin and gut microbiomes, and their vertical transmission, remains incomplete as major lineages and varied forms of parental care remain unexplored. The diverse and elaborate forms of parental care exhibited by amphibians constitute an ideal system to study microbe transmission, yet investigations of vertical transmission among frogs and salamanders have been inconclusive. In this study, we assess bacteria transmission in Herpele squalostoma, an oviparous direct-developing caecilian in which females obligately attend juveniles that feed on their mother's skin (dermatophagy). RESULTS: We used 16S rRNA amplicon-sequencing of the skin and gut of wild caught H. squalostoma individuals (males, females, including those attending juveniles) as well as environmental samples. Sourcetracker analyses revealed that juveniles obtain an important portion of their skin and gut bacteria communities from their mother. The contribution of a mother's skin to the skin and gut of her respective juveniles was much larger than that of any other bacteria source. In contrast to males and females not attending juveniles, only the skins of juveniles and their mothers were colonized by bacteria taxa Verrucomicrobiaceae, Nocardioidaceae, and Erysipelotrichaceae. In addition to providing indirect evidence for microbiome transmission linked to parental care among amphibians, our study also points to noticeable differences between the skin and gut communities of H. squalostoma and that of many frogs and salamanders, which warrants further investigation. CONCLUSION: Our study is the first to find strong support for vertical bacteria transmission attributed to parental care in a direct-developing amphibian species. This suggests that obligate parental care may promote microbiome transmission in caecilians.

Once a reservoir, always a reservoir? Seasonality affects the pathogen maintenance potential of amphibian hosts.

Host species that can independently maintain a pathogen in a host community and contribute to infection in other species are important targets for disease management. However, the potential of host species to maintain a pathogen is not fixed over time, and an important challenge is understanding how within- and across-season variability in host maintenance potential affects pathogen persistence over longer time scales relevant for disease management (e.g., years). Here, we sought to understand the causes and consequences of seasonal infection dynamics in leopard frogs (Rana sphenocephala and Rana pipiens) infected with the fungal pathogen Batrachochytrium dendrobatidis (Bd). We addressed three questions broadly applicable to seasonal host-parasite systems. First, to what degree are observed seasonal patterns in infection driven by temperature-dependent infection processes compared to seasonal host demographic processes? Second, how does seasonal variation in maintenance potential affect long-term pathogen persistence in multi-host communities? Third, does high deterministic maintenance potential relate to the long-term stochastic persistence of pathogens in host populations with seasonal infection dynamics? To answer these questions, we used field data collected over 3 years on >1400 amphibians across four geographic locations, laboratory and mesocosm experiments, and a novel mathematical model. We found that the mechanisms that drive seasonal prevalence were different from those driving seasonal infection intensity. Seasonal variation in Bd prevalence was driven primarily by changes in host contact rates associated with breeding migrations to and from aquatic habitat. In contrast, seasonal changes in infection intensity were driven by temperature-induced changes in Bd growth rate. Using our model, we found that the maintenance potential of leopard frogs varied significantly throughout the year and that seasonal troughs in infection prevalence made it unlikely that leopard frogs were responsible for long-term Bd persistence in these seasonal amphibian communities, highlighting the importance of alternative pathogen reservoirs for Bd persistence. Our results have broad implications for management in seasonal host-pathogen systems, showing that seasonal changes in host and pathogen vital rates, rather than the depletion of susceptible hosts, can lead to troughs in pathogen prevalence and stochastic pathogen extirpation.

Batrachochytrium salamandrivorans ELICITS ACUTE STRESS RESPONSE IN SPOTTED SALAMANDERS BUT NOT INFECTION OR MORTALITY.

The emerging fungal pathogen Batrachochytrium salamandrivorans (Bsal) is a major threat to amphibian species worldwide with potential to infect many species if it invades salamander biodiversity hotspots in the Americas. Bsal can cause the disease chytridiomycosis, and it is important to assess the risk of Bsal-induced chytridiomycosis to species in North America. We evaluated the susceptibility to Bsal of the common and widespread spotted salamander, Ambystoma maculatum, across life history stages and monitored the effect of Bsal exposure on growth rate and response of the stress hormone, corticosterone. We conclude that spotted salamanders appear resistant to Bsal because they showed no indication of disease or infection, and experienced minor effects on growth upon exposure. While we focused on a single population for this study, results were consistent across conditions of exposure including high or repeated doses of Bsal, life-stage at exposure, environmental conditions including two temperatures and two substrates, and promoting pathogen infectivity by conditioning Bsal cultures with thyroid hormone. Exposure to high levels of Bsal elicited an acute but not chronic increase in corticosterone in spotted salamanders, and reduced growth. We hypothesize that the early acute increase in corticosterone facilitated mounting an immune response to the pathogen, perhaps through immunoredistribution to the skin, but further study is needed to determine immune responses to Bsal. These results will contribute to development of appropriate Bsal management plans to conserve species at risk of emerging disease.

Publisher Correction: Host-associated microbiomes are predicted by immune system complexity and climate.

Following publication of the original paper [1], it was reported that an error in the processing of Fig. 8 occurred. In the online HTML version of the article, Fig. 8 was presented as a duplication of Fig. 7. The original article [1] has been corrected.

Host-associated microbiomes are predicted by immune system complexity and climate.

BACKGROUND: Host-associated microbiomes, the microorganisms occurring inside and on host surfaces, influence evolutionary, immunological, and ecological processes. Interactions between host and microbiome affect metabolism and contribute to host adaptation to changing environments. Meta-analyses of host-associated bacterial communities have the potential to elucidate global-scale patterns of microbial community structure and function. It is possible that host surface-associated (external) microbiomes respond more strongly to variations in environmental factors, whereas internal microbiomes are more tightly linked to host factors. RESULTS: Here, we use the dataset from the Earth Microbiome Project and accumulate data from 50 additional studies totaling 654 host species and over 15,000 samples to examine global-scale patterns of bacterial diversity and function. We analyze microbiomes from non-captive hosts sampled from natural habitats and find patterns with bioclimate and geophysical factors, as well as land use, host phylogeny, and trophic level/diet. Specifically, external microbiomes are best explained by variations in mean daily temperature range and precipitation seasonality. In contrast, internal microbiomes are best explained by host factors such as phylogeny/immune complexity and trophic level/diet, plus climate. CONCLUSIONS: Internal microbiomes are predominantly associated with top-down effects, while climatic factors are stronger determinants of microbiomes on host external surfaces. Host immunity may act on microbiome diversity through top-down regulation analogous to predators in non-microbial ecosystems. Noting gaps in geographic and host sampling, this combined dataset represents a global baseline available for interrogation by future microbial ecology studies.

Survey of Pathogenic Chytrid Fungi (Batrachochytrium dendrobatidis and B. salamandrivorans) in Salamanders from Three Mountain Ranges in Europe and the Americas.

Batrachochytrium salamandrivorans (Bsal) is a virulent fungal pathogen that infects salamanders. It is implicated in the recent collapse of several populations of fire salamanders in Europe. This pathogen seems much like that of its sister species, Batrachochytrium dendrobatidis (Bd), the agent responsible for anuran extinctions and extirpations worldwide, and is considered to be an emerging global threat to salamander communities. Bsal thrives at temperatures found in many mountainous regions rich in salamander species; because of this, we have screened specimens of salamanders representing 17 species inhabiting mountain ranges in three continents: The Smoky Mountains, the Swiss Alps, and the Peruvian Andes. We screened 509 salamanders, with 192 representing New World salamanders that were never tested for Bsal previously. Bsal was not detected, and Bd was mostly present at low prevalence except for one site in the Andes.

Co-Infection by Chytrid Fungus and Ranaviruses in Wild and Harvested Frogs in the Tropical Andes.

While global amphibian declines are associated with the spread of Batrachochytrium dendrobatidis (Bd), undetected concurrent co-infection by other pathogens may be little recognized threats to amphibians. Emerging viruses in the genus Ranavirus (Rv) also cause die-offs of amphibians and other ectotherms, but the extent of their distribution globally, or how co-infections with Bd impact amphibians are poorly understood. We provide the first report of Bd and Rv co-infection in South America, and the first report of Rv infections in the amphibian biodiversity hotspot of the Peruvian Andes, where Bd is associated with extinctions. Using these data, we tested the hypothesis that Bd or Rv parasites facilitate co-infection, as assessed by parasite abundance or infection intensity within individual adult frogs. Co-infection occurred in 30% of stream-dwelling frogs; 65% were infected by Bd and 40% by Rv. Among terrestrial, direct-developing Pristimantis frogs 40% were infected by Bd, 35% by Rv, and 20% co-infected. In Telmatobius frogs harvested for the live-trade 49% were co-infected, 92% were infected by Bd, and 53% by Rv. Median Bd and Rv loads were similar in both wild (Bd = 101.2 Ze, Rv = 102.3 viral copies) and harvested frogs (Bd = 103.1 Ze, Rv = 102.7 viral copies). While neither parasite abundance nor infection intensity were associated with co-infection patterns in adults, these data did not include the most susceptible larval and metamorphic life stages. These findings suggest Rv distribution is global and that co-infection among these parasites may be common. These results raise conservation concerns, but greater testing is necessary to determine if parasite interactions increase amphibian vulnerability to secondary infections across differing life stages, and constitute a previously undetected threat to declining populations. Greater surveillance of parasite interactions may increase our capacity to contain and mitigate the impacts of these and other wildlife diseases.