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  1. Abstract

    Variable harlequin frogsAtelopus variushave declined significantly throughout their range as a result of infection with the fungal pathogenBatrachochytrium dendrobatidis(Bd). The Panama Amphibian Rescue and Conservation Project maintains an ex situ population of this Critically Endangered species. We conducted a release trial with surplus captive-bredA. variusindividuals to improve our ability to monitor frog populations post-release, observe dispersal patterns after freeing them into the wild and learn about threats to released frogs, as well as to determine whether natural skin toxin defences of frogs could be restored inside mesocosms in the wild and to compare Bd dynamics in natural amphibian communities at the release site vs a non-release site. The 458 released frogs dispersed rapidly and were difficult to re-encounter unless they carried a radio transmitter. No frog was seen after 36 days following release. Thirty frogs were fitted with radio transmitters and only half were trackable by day 10. Tetrodotoxin was not detected in the skins of the frogs inside mesocosms for up to 79 days. Bd loads in other species present at sites were high prior to release and decreased over time in a pattern probably driven by weather. No differences were observed in Bd prevalence between the release and non-release sites. This trial showed that refinements of our methods and approaches are required to study captiveAtelopusfrogs released into wild conditions. We recommend continuing release trials of captive-bred frogs with post-release monitoring methods, using an adaptive management framework to advance the field of amphibian reintroduction ecology.

     
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    Free, publicly-accessible full text available May 1, 2025
  2. Abstract

    Mosquitoes are a complex nuisance around the world and tropical countries bear the brunt of the burden of mosquito-borne diseases. Rwanda has had success in reducing malaria and some arboviral diseases over the last few years, but still faces challenges to elimination. By building our understanding of in situ mosquito communities in Rwanda at a disturbed, human-occupied site and at a natural, preserved site, we can build our understanding of natural mosquito microbiomes toward the goal of implementing novel microbial control methods. Here, we examined the composition of collected mosquitoes and their microbiomes at two diverse sites using Cytochrome c Oxidase I sequencing and 16S V4 high-throughput sequencing. The majority (36 of 40 species) of mosquitoes captured and characterized in this study are the first-known record of their species for Rwanda but have been characterized in other nations in East Africa. We found significant differences among mosquito genera and among species, but not between mosquito sexes or catch method. Bacteria of interest for arbovirus control,Asaia,Serratia, andWolbachia, were found in abundance at both sites and varied greatly by species.

     
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  3. Kalendar, Ruslan (Ed.)

    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.

     
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  4. Bats are widespread mammals that play key roles in ecosystems as pollinators and insectivores. However, there is a paucity of information about bat-associated microbes, in particular their fungal communities, despite the important role microbes play in host health and overall host function. The emerging fungal disease, white-nose syndrome, presents a potential challenge to the bat microbiome and understanding healthy bat-associated taxa will provide valuable information about potential microbiome-pathogen interactions. To address this knowledge gap, we collected 174 bat fur/skin swabs from 14 species of bats captured in five locations in New Mexico and Arizona and used high-throughput sequencing of the fungal internal transcribed (ITS) region to characterize bat-associated fungal communities. Our results revealed a highly heterogeneous bat mycobiome that was structured by geography and bat species. Furthermore, our data suggest that bat-associated fungal communities are affected by bat foraging, indicating the bat skin microbiota is dynamic on short time scales. Finally, despite the strong effects of site and species, we found widespread and abundant taxa from several taxonomic groups including 
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  5. The amphibian chytrid fungus, Batrachochytrium salamandrivorans ( Bsal ) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 
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  6. The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species ( Xenopus laevis ) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis , they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 
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  7. With emerging diseases on the rise, there is an urgent need to identify and understand novel mechanisms of prophylactic protection in vertebrate hosts. Inducing resistance against emerging pathogens through prophylaxis is an ideal management strategy that may impact pathogens and their host-associated microbiome. The host microbiome is recognized as a critical component of immunity, but the effects of prophylactic inoculation on the microbiome are unknown. In this study, we investigate the effects of prophylaxis on host microbiome composition, focusing on the selection of anti-pathogenic microbes contributing to host acquired immunity in a model host–fungal disease system, amphibian chytridiomycosis. We inoculated larval Pseudacris regilla against the fungal pathogen Batrachochytrium dendrobatidis ( Bd ) with a Bd metabolite-based prophylactic. Increased prophylactic concentration and exposure duration were associated with significant increases in proportions of putatively Bd -inhibitory host-associated bacterial taxa, indicating a protective prophylactic-induced shift towards microbiome members that are antagonistic to Bd. Our findings are in accordance with the adaptive microbiome hypothesis, where exposure to a pathogen alters the microbiome to better cope with subsequent pathogen encounters. Our study advances research on the temporal dynamics of microbiome memory and the role of prophylaxis-induced shifts in microbiomes contributing to prophylaxis effectiveness. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 
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  8. The amphibian skin microbiome is an important component of anti-pathogen defense, but the impact of environmental change on the link between microbiome composition and host stress remains unclear. In this study, we used radiotelemetry and host translocation to track microbiome composition and function, pathogen infection, and host stress over time across natural movement paths for the forest-associated treefrog, Boana faber. We found a negative correlation between cortisol levels and putative microbiome function for frogs translocated to forest fragments, indicating strong integration of host stress response and anti-pathogen potential of the microbiome. Additionally, we observed a capacity for resilience (resistance to structural change and functional loss) in the amphibian skin microbiome, with maintenance of putative pathogen-inhibitory function despite major temporal shifts in microbiome composition. Although microbiome community composition did not return to baseline during the study period, the rate of microbiome change indicated that forest fragmentation had more pronounced effects on microbiome composition than translocation alone. Our findings reveal associations between stress hormones and host microbiome defenses, with implications for resilience of amphibians and their associated microbes facing accelerated tropical deforestation. 
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  9. Abstract Batrachochytrium salamandrivorans ( Bsal ) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal , and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America. 
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