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

   https://doi.org/10.1111/1365-2656.13386  

   Le Sage, Emily H. ; LaBumbard, Brandon C. ; Reinert, Laura K. ; Miller, Brian T. ; Richards‐Zawacki, Corinne L. ; Woodhams, Doug C. ; Rollins‐Smith, Louise A. ; Bielby, ed., Jon ( November 2020 , Journal of Animal Ecology)

   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 againstBdcorrespond 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 ofBdgrowth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibitBdgrowth in vitro were used as an estimate of predicted anti‐Bdfunction of the skin microbiome.

   Batrachochytrium dendrobatidisdynamics followed the expected seasonal fluctuations—peaks in cooler months—which coincided with when host mucosal defences were most potent againstBd. Specifically, the concentration and expression of stored AMPs cycled synchronously withBddynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibitBdgrowth was greatest when risk ofBdinfection was highest.

   We interpret the increase in peptide storage in the fall and the shift to a more anti‐Bdmicrobiome 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 ofBdin this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.

    

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3. Isolation and maintenance of Batrachochytrium salamandrivorans cultures

   https://doi.org/10.3354/dao03488  

   Robinson, KA ; Pereira, KE ; Bletz, MC ; Carter, ED ; Gray, MJ ; Piovia-Scott, J ; Romansic, JM ; Woodhams, DC ; Fritz-Laylin, L ( June 2020 , Diseases of Aquatic Organisms)

   null (Ed.)

   Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans ( Bsal ) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis ( Bd ), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal . Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue. 

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4. Seasonality of host immunity in a tropical disease system

   https://doi.org/10.1002/ecs2.4158  

   Rosa, Gonçalo M. ; Perez, Rachel ; Richards, Lora A. ; Richards‐Zawacki, Corinne L. ; Smilanich, Angela M. ; Reinert, Laura K. ; Rollins‐Smith, Louise A. ; Wetzel, Daniel P. ; Voyles, Jamie ( July 2022 , Ecosphere)

   Infectious disease systems frequently exhibit strong seasonal patterns, yet the mechanisms that underpin intra‐annual cycles are unclear, particularly in tropical regions. We hypothesized that host immune function fluctuates seasonally, contributing to oscillations in infection patterns in a tropical disease system. To test this hypothesis, we investigated a key host defense of amphibians against a lethal fungal pathogen,Batrachochytrium dendrobatidis(Bd). We integrated two field experiments in which we perturbed amphibian skin secretions, a critical host immune mechanism, in Panamanian rocket frogs (Colostethus panamansis). We found that this immunosuppressive technique of reducing skin secretions in wild frog populations increasedBdprevalence and infection intensity, indicating that this immune defense contributes to resistance toBdin wild frog populations. We also found that the chemical composition and anti‐Bdeffectiveness of frog skin secretions varied across seasons, with greater pathogen inhibition during the dry season relative to the wet season. These results suggest that the effectiveness of this host defense mechanism shifts across seasons, likely contributing to seasonal infection patterns in a lethal disease system. More broadly, our findings indicate that host immune defenses can fluctuate across seasons, even in tropical regions where temperatures are relatively stable, which advances our understanding of intra‐annual cycles of infectious disease dynamics.

    

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5. When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis

   https://doi.org/10.1093/icb/icac060  

   Gass, Jordan ; Voyles, Jamie ( May 2022 , Integrative and Comparative Biology)

   To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

    

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