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

   https://doi.org/10.1128/iai.00020-22  

   Rollins-Smith, Louise A. ; Reinert, Laura K. ; Le Sage, Mitchell ; Linney, Kaitlyn N. ; Gillard, Bria M. ; Umile, Thomas P. ; Minbiole, Kevin P. ( March 2022 , Infection and Immunity)

   Noverr, Mairi C. (Ed.)

   ABSTRACT 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. 

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2. High-efficiency electroporation of chytridfungi

   https://doi.org/10.1038/s41598-020-71618-2  

   Swafford, Andrew J. ; Hussey, Shane P. ; Fritz-Laylin, Lillian K. ( December 2020 , Scientific Reports)

   null (Ed.)

   Abstract Two species of parasitic fungi from the phylum Chytridiomycota (chytrids)are annihilating global amphibian populations. These chytrid species— Batrachochytrium dendrobatidis and B. salamandrivorans —have high rates of mortality and transmission. Uponestablishing infection in amphibians, chytrids rapidly multiply within the skin anddisrupt their hosts’ vital homeostasis mechanisms. Current disease models suggest thatchytrid fungi locate and infect their hosts during a motile, unicellular ‘zoospore’ lifestage. Moreover, other chytrid species parasitize organisms from across the tree oflife, making future epidemics in new hosts a likely possibility. Efforts to mitigate thedamage and spread of chytrid disease have been stymied by the lack of knowledge aboutbasic chytrid biology and tools with which to test molecular hypotheses about diseasemechanisms. To overcome this bottleneck, we have developed high-efficiency delivery ofmolecular payloads into chytrid zoospores using electroporation. Our electroporationprotocols result in payload delivery to between 75 and 97% of living cells of threespecies: B. dendrobatidis, B. salamandrivorans, and anon-pathogenic relative, Spizellomyces punctatus .This method lays the foundation for molecular genetic tools needed to establishecological mitigation strategies and answer broader questions in evolutionary and cellbiology. 

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

   https://doi.org/10.1128/aem.01818-21  

   Jiménez, Randall R. ; Carfagno, Amy ; Linhoff, Luke ; Gratwicke, Brian ; Woodhams, Douglas C. ; Chafran, Liana Soares ; Bletz, Molly C. ; Bishop, Barney ; Muletz-Wolz, Carly R. ( April 2022 , Applied and Environmental Microbiology)

   Reguera, Gemma (Ed.)

   ABSTRACT 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. 

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4. Preparing for invasion: Assessing risk of infection by chytrid fungi in southeastern plethodontid salamanders

   https://doi.org/10.1002/jez.2427  

   Friday, Brenna ; Holzheuser, Chace ; Lips, Karen R. ; Longo, Ana V. ( November 2020 , Journal of Experimental Zoology Part A: Ecological and Integrative Physiology)

   Understanding the responses of naïve communities to the invasion of multihost pathogens requires accurate estimates of susceptibility across taxa. In the Americas, the likely emergence of a second amphibian pathogenic fungus (Batrachochytrium salamandrivorans, Bsal) calls for new ways of prioritizing disease mitigation among species due to the high diversity of naïve hosts with priorB. dendrobatidis(Bd) infections. Here, we applied the concept of pathogenic potential to quantify the virulence of chytrid fungi on naïve amphibians and evaluate species for conservation efforts in the event of an outbreak. The benefit of this measure is that it combines and summarizes the variation in disease effects into a single numerical index, allowing for comparisons across species, populations or groups of individuals that may inherently exhibit differences in susceptibility. As a proof of concept, we obtained standardized responses of disease severity by performing experimental infections withBsalon five plethodontid salamanders from southeastern United States. Four out of five species carried natural infections ofBdat the start of the experiments. We showed thatBsalexhibited its highest value of pathogenic potential in a species that is already declining (Desmognathus auriculatus). We find that this index provides additional information beyond the standard measures of disease prevalence, intensity, and mortality, because it leveraged these disease parameters within each categorical group. Scientists and practitioners could use this measure to justify research, funding, trade, or conservation measures.

    

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5. Spatiotemporal and ontogenetic variation, microbial selection, and predicted Bd-inhibitory function in the skin-associated microbiome of a Rocky Mountain amphibian

   https://doi.org/10.3389/fmicb.2022.1020329  

   Goodwin, Kenen B. ; Hutchinson, Jaren D. ; Gompert, Zachariah ( December 2022 , Frontiers in Microbiology)

   Host-associated microbiomes play important roles in host health and pathogen defense. In amphibians, the skin-associated microbiota can contribute to innate immunity with potential implications for disease management. Few studies have examined season-long temporal variation in the amphibian skin-associated microbiome, and the interactions between bacteria and fungi on amphibian skin remain poorly understood. We characterize season-long temporal variation in the skin-associated microbiome of the western tiger salamander ( Ambystoma mavortium ) for both bacteria and fungi between sites and across salamander life stages. Two hundred seven skin-associated microbiome samples were collected from salamanders at two Rocky Mountain lakes throughout the summer and fall of 2018, and 127 additional microbiome samples were collected from lake water and lake substrate. We used 16S rRNA and ITS amplicon sequencing with Bayesian Dirichlet-multinomial regression to estimate the relative abundances of bacterial and fungal taxa, test for differential abundance, examine microbial selection, and derive alpha diversity. We predicted the ability of bacterial communities to inhibit the amphibian chytrid fungus Batrachochytrium dendrobatidis ( Bd ), a cutaneous fungal pathogen, using stochastic character mapping and a database of Bd -inhibitory bacterial isolates. For both bacteria and fungi, we observed variation in community composition through time, between sites, and with salamander age and life stage. We further found that temporal trends in community composition were specific to each combination of salamander age, life stage, and lake. We found salamander skin to be selective for microbes, with many taxa disproportionately represented relative to the environment. Salamander skin appeared to select for predicted Bd -inhibitory bacteria, and we found a negative relationship between the relative abundances of predicted Bd -inhibitory bacteria and Bd . We hope these findings will assist in the conservation of amphibian species threatened by chytridiomycosis and other emerging diseases. 

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