The impacts of water quality on the amphibian chytrid fungal pathogen: A systematic review.

The pathogenic fungus Batrachochytrium dendrobatidis has caused declines of amphibians worldwide. Yet our understanding of how water quality influences fungal pathogenicity is limited. Here, we reviewed experimental studies on the effect of water quality on this pathogen to determine which parameters impacted disease dynamics consistently. The strongest evidence for protective effects is salinity which shows strong antifungal properties in hosts at natural levels. Although many fungicides had detrimental effects on the fungal pathogen in vitro, their impact on the host is variable and they can worsen infection outcomes. However, one fungicide, epoxiconazole, reduced disease effects experimentally and likely in the field. While heavy metals are frequently studied, there is weak evidence that they influence infection outcomes. Nitrogen and phosphorous do not appear to impact pathogen growth or infection in the amphibian host. The effects of other chemicals, like pesticides and disinfectants on infection were mostly unclear with mixed results or lacking an in vivo component. Our study shows that water chemistry does impact disease dynamics, but the effects of specific parameters require more investigation. Improving our understanding of how water chemistry influences disease dynamics will help predict the impact of chytridiomycosis, especially in amphibian populations affected by land use changes.

Instant killing of pathogenic chytrid fungi by disposable nitrile gloves prevents disease transmission between amphibians.

To prevent transmission of the pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), hygiene protocols prescribe the single use of disposable gloves for handling amphibians. We discovered that rinse water from nitrile gloves instantly kills 99% of Bd and Bsal zoospores. Transmission experiments using midwife toads (Alytes obstetricans) and Bd, and Alpine newts (Ichthyosaura alpestris) and Bsal, show that the use of the same pair of gloves for 2 subsequent individuals does not result in significant transmission of any chytrid fungus. In contrast, handling infected amphibians bare-handed caused transmission of Bsal in 4 out of 10 replicates, but did not result in transmission of Bd. Based on the manufacturer's information, high resolution mass spectrometry (HRMS) and colorimetric tests, calcium lactate and calcium nitrate were identified as compounds with antifungal activity against both Bd and Bsal. These findings corroborate the importance of wearing gloves as an important sanitary measure in amphibian disease prevention. If the highly recommended single use of gloves is not possible, handling multiple post-metamorphic amphibians with the same pair of nitrile gloves should still be preferred above bare-handed manipulation.

Investigating the potential use of an ionic liquid (1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide) as an anti-fungal treatment against the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

The disease chytridiomycosis, caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to global amphibian declines. Bd infects the keratinized epidermal tissue in amphibians and causes hyperkeratosis and excessive skin shedding. In individuals of susceptible species, the regulatory function of the amphibian's skin is disrupted resulting in an electrolyte depletion, osmotic imbalance, and eventually death. Safe and effective treatments for chytridiomycosis are urgently needed to control chytrid fungal infections and stabilize populations of endangered amphibian species in captivity and in the wild. Currently, the most widely used anti-Bd treatment is itraconazole. Preparations of itraconazole formulated for amphibian use has proved effective, but treatment involves short baths over seven to ten days, a process which is logistically challenging, stressful, and causes long-term health effects. Here, we explore a novel anti-fungal therapeutic using a single application of the ionic liquid, 1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BMP-NTf2), for the treatment of chytridiomycosis. BMP-NTf2 was found be effective at killing Bd in vitro at low concentrations (1:1000 dilution). We tested BMP-NTf2 in vivo on two amphibian species, one that is relatively tolerant of chytridiomycosis (Pseudacris regilla) and one that is highly susceptible (Dendrobates tinctorius). A toxicity trial revealed a surprising interaction between Bd infection status and the impact of BMP-NTf2 on D. tinctorius survival. Uninfected D. tinctorius tolerated BMP-NTf2 (mean ± SE; 96.01 ± 9.00 µl/g), such that only 1 out of 30 frogs died following treatment (at a dose of 156.95 µL/g), whereas, a lower dose (mean ± SE; 97.45 ± 3.52 µL/g) was not tolerated by Bd-infected D. tinctorius, where 15 of 23 frogs died shortly upon BMP-NTf2 application. Those that tolerated the BMP-NTf2 application did not exhibit Bd clearance. Thus, BMP-NTf2 application, under the conditions tested here, is not a suitable option for clearing Bd infection in D. tinctorius. However, different results were obtained for P. regilla. Two topical applications of BMP-NTf2 on Bd-infected P. regilla (using a lower BMP-NTf2 dose than on D. tinctorius, mean ± SE; 9.42 ± 1.43 µL/g) reduced Bd growth, although the effect was lower than that obtained by daily doses of itracanozole (50% frogs exhibited complete clearance on day 16 vs. 100% for itracanozole). Our findings suggest that BMP-NTf2 has the potential to treat Bd infection, however the effect depends on several parameters. Further optimization of dose and schedule are needed before BMP-NTf2 can be considered as a safe and effective alternative to more conventional antifungal agents, such as itraconazole.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Actin guides filamentous rhizoid growth and morphogenesis in the zoosporic fungus Chytriomyces hyalinus.

The advantage of filamentous growth to the fungal lifestyle is so great that it arose multiple times. Most zoosporic fungi from phylum Chytridiomycota exhibit a monocentric thallus form consisting of anucleate filamentous rhizoids that anchor reproductive sporangia to substrata and absorb nutrients. Actin function during polarized growth and cytokinesis is well documented across eukaryotes, but its role in sculpting nonhyphal, nonyeast fungal cells is unknown. We sought to provide a basis for comparing actin organization among major fungal lineages and to investigate the effects of actin disruption on morphogenesis in a monocentric thallus. Using fluorescence microscopy, we observed fixed, rhodamine phalloidin-stained actin in chemically fixed Chytriomyces hyalinus, exemplifying monocentric thallus development within the diverse, zoosporic phylum Chytridiomycota. We also compared rhizoid lengths and rhizoid branching of thalli incubated with the actin inhibitor latrunculin B to determine the effects of actin disruption on morphology. Actin was concentrated at the tips of growing rhizoids. Actin cables typically formed cortical, parallel arrays in hyphae, but in mature sporangia they were concentrated in a funnel-shaped array in the central region. Thalli treated with latrunculin B had shorter rhizoids with fewer branches than controls. In both hyphae and monocentric thalli, actin localization coincides with active, polarized growth and cytokinesis. Specific actin localization patterns are largely shared between monocentric species but differ significantly from patterns observed in hyphae. Actin integrity is critical for sustaining filamentous growth in all fungi.

Disease Exposure and Antifungal Bacteria on Skin of Invasive Cane Toads, Australia.

Cane toads, an invasive species in Australia, are resistant to fungal pathogens affecting frogs worldwide (Batrachochytrium dendrobatidis). From toad skin swabs, we detected higher proportions of bacteria with antifungal properties in Queensland, where toad and pathogen distributions overlap, than in other sites. This finding suggests that site-specific pathogen pressures help shape skin microbial communities.

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.

Integrating the role of antifungal bacteria into skin symbiotic communities of three Neotropical frog species.

Chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), has led to population declines and extinctions of frog species around the world. While it is known that symbiotic skin bacteria can play a protective role against pathogens, it is not known how these defensive bacteria are integrated into the bacterial community on amphibian skin. In this study, we used 16S rRNA gene amplicon sequencing, culturing and Bd inhibition bioassays to characterize the communities of skin bacteria on three Neotropical frog species that persist in a Bd-infected area in Panama and determined the abundance and integration of anti-Bd bacteria into the community. We found that the two treefrog species had a similar bacterial community structure, which differed from the more diverse community found on the terrestrial frog. Co-occurrence networks also revealed differences between frog species such that the treefrogs had a significantly higher number of culturable Bd-inhibitory OTUs with high centrality scores compared with the terrestrial frog. We found that culture-dependent OTUs captured between 21 and 39% of the total relative abundance revealed in culture-independent communities. Our results suggest different ecological strategies occurring within skin antifungal communities on host species that have not succumbed to Bd infections in the wild.

Viscosin-like lipopeptides from frog skin bacteria inhibit Aspergillus fumigatus and Batrachochytrium dendrobatidis detected by imaging mass spectrometry and molecular networking.

Amphibian populations worldwide have declined and in some cases become extinct due to chytridiomycosis, a pandemic disease caused by the fungus Batrachochytrium dendrobatidis; however, some species have survived these fungal epidemics. Previous studies have suggested that the resistance of these species is due to the presence of cutaneous bacteria producing antifungal metabolites. As our understanding of these metabolites is still limited, we assessed the potential of such compounds against human-relevant fungi such as Aspergillus. In this work we isolated 201 bacterial strains from fifteen samples belonging to seven frog species collected in the highlands of Panama and tested them against Aspergillus fumigatus. Among the 29 bacterial isolates that exhibited antifungal activity, Pseudomonas cichorii showed the greatest inhibition. To visualize the distribution of compounds and identify them in the inhibition zone produced by P. cichorii, we employed MALDI imaging mass spectrometry (MALDI IMS) and MS/MS molecular networking. We identified viscosin and massetolides A, F, G and H in the inhibition zone. Furthermore, viscosin was isolated and evaluated in vitro against A. fumigatus and B. dendrobatidis showing MIC values of 62.50 µg/mL and 31.25 µg/mL, respectively. This is the first report of cyclic depsipeptides with antifungal activity isolated from frog cutaneous bacteria.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Skin glands of an aquatic salamander vary in size and distribution and release antimicrobial secretions effective against chytrid fungal pathogens.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in an aquatic salamander, the three-toed amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.

Antifungal efficacy of F10SC veterinary disinfectant against Batrachochytrium dendrobatidis.

The Infectious disease chytridiomycosis, which is caused by the fungal pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of amphibian declines and extinction. In vitro B. dendrobatidis is susceptible to a range of disinfectants, but not all have been tested on animals and some that have been proven effective have harmful side effects on the surrounding environment or the animals being treated. We tested the efficacy of F10SC veterinary disinfectant to treat B. dendrobatidis in experimentally infected tadpole and juvenile Sclerophrys gutturalis and tadpoles of Sclerophrys poweri and Amietia hymenopus. The minimum inhibitory concentration for F10SC on in vitro B. dendrobatidis ranged between 1:7000 for 5-min contact time and 1:10000 for 10-min contact time. Based on the survival data of test animals the no observed effect concentration for 15-min contact time was estimated to be 1:2000 dilution for juveniles, and 1:10000 for tadpoles. In S. gutturalis juveniles an 86% infection clearance rate was achieved after five 15-min doses of 1:3000 dilution. A 100% clearance was achieved in A. hymenopus tadpoles after seven 15-min doses of 1:10000 dilution, and after nine doses of the same treatment in S. poweri tadpoles. F10SC has the benefit of being a concentrated compound that provides a treatment protocol which is nontoxic to tadpoles and post-metamorphic individuals, has a short half-life and is effective against B. dendrobatidis during short contact times, but further testing on different species of amphibians is advised.

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.

Efficacy of chemical disinfectants for the containment of the salamander chytrid fungus Batrachochytrium salamandrivorans.

The recently emerged chytrid fungus Batrachochytrium salamandrivorans (Bsal) causes European salamander declines. Proper hygiene protocols including disinfection procedures are crucial to prevent disease transmission. Here, the efficacy of chemical disinfectants in killing Bsal was evaluated. At all tested conditions, Biocidal®, Chloramine-T®, Dettol medical®, Disolol®, ethanol, F10®, Hibiscrub®, potassium permanganate, Safe4®, sodium hypochlorite, and Virkon S®, were effective at killing Bsal. Concentrations of 5% sodium chloride or lower, 0.01% peracetic acid and 0.001-1% copper sulphate were inactive against Bsal. None of the conditions tested for hydrogen peroxide affected Bsal viability, while it did kill Batrachochytrium dendrobatidis (Bd). For Bsal, enzymatic breakdown of hydrogen peroxide by catalases and specific morphological features (clustering of sporangia, development of new sporangia within the original sporangium), were identified as fungal factors altering susceptibility to several of the disinfectants tested. Based on the in vitro results we recommend 1% Virkon S®, 4% sodium hypochlorite and 70% ethanol for disinfecting equipment in the field, lab or captive setting, with a minimal contact time of 5 minutes for 1% Virkon S® and 1 minute for the latter disinfectants. These conditions not only efficiently target Bsal, but also Bd and Ranavirus.

A pesticide paradox: fungicides indirectly increase fungal infections.

There are many examples where the use of chemicals have had profound unintended consequences, such as fertilizers reducing crop yields (paradox of enrichment) and insecticides increasing insect pests (by reducing natural biocontrol). Recently, the application of agrochemicals, such as agricultural disinfectants and fungicides, has been explored as an approach to curb the pathogenic fungus, Batrachochytrium dendrobatidis (Bd), which is associated with worldwide amphibian declines. However, the long-term, net effects of early-life exposure to these chemicals on amphibian disease risk have not been thoroughly investigated. Using a combination of laboratory experiments and analysis of data from the literature, we explored the effects of fungicide exposure on Bd infections in two frog species. Extremely low concentrations of the fungicides azoxystrobin, chlorothalonil, and mancozeb were directly toxic to Bd in culture. However, estimated environmental concentrations of the fungicides did not reduce Bd on Cuban tree frog (Osteopilus septentrionalis) tadpoles exposed simultaneously to any of these fungicides and Bd, and fungicide exposure actually increased Bd-induced mortality. Additionally, exposure to any of these fungicides as tadpoles resulted in higher Bd abundance and greater Bd-induced mortality when challenged with Bd post-metamorphosis, an average of 71 d after their last fungicide exposure. Analysis of data from the literature revealed that previous exposure to the fungicide itraconazole, which is commonly used to clear Bd infections, made the critically endangered booroolong frog (Litoria booroolongensis) more susceptible to Bd. Finally, a field survey revealed that Bd prevalence was positively associated with concentrations of fungicides in ponds. Although fungicides show promise for controlling Bd, these results suggest that, if fungicides do not completely eliminate Bd or if Bd recolonizes, exposure to fungicides has the potential to do more harm than good. To ensure that fungicide applications have the intended consequence of curbing amphibian declines, researchers must identify which fungicides do not compromise the pathogen resistance mechanisms of amphibians.

Microplastics alter composition of fungal communities in aquatic ecosystems.

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.

THE PHARMACOKINETICS OF TOPICAL ITRACONAZOLE IN PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI).

Chytridiomycosis is caused by the fungus Batrachochytrium dendrobatidis and is one of the primary causes of the global decline in amphibian populations and specifically of the Panamanian golden frog ( Atelopus zeteki ). Itraconazole has been demonstrated to be an effective treatment for chytridiomycosis by inhibiting cytochrome P450, a major enzyme important for the structure of B. dendrobatidis zoospores' plasma membranes. However, anecdotal reports of toxicity in this and other amphibian species have been reported at the 0.01% concentration. This study is the first to determine pharmacokinetics of 0.01% and 0.001% itraconazole in the Panamanian golden frog. Frogs were bathed 10 min, euthanized, and skin, liver, and heart were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 36 hr. Itraconazole concentrations were measured using high performance liquid chromatography, and the minimum inhibitory concentration (MIC) of itraconazole (0.032 µg/ml) for B. dendrobatidis was used to determine whether therapeutic concentrations were attained. Itraconazole was detected in all tissues at both concentrations, indicating systemic absorption. At the 0.01% itraconazole bath, itraconazole concentrations in all tissues exceeded the MIC at all time points, and the lack of decline until the end of the study at 36 hr precluded determining a disappearance half-life. With the 0.001% bath, itraconazole exceeded the MIC and declined with a disappearance half-life that markedly varied (14.1-1,244 min). This study augments the growing literature base on chytridiomycosis and seeks to aid in further experimental attempts to find the most-optimal treatment protocol for this disease.

Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen.

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides-the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

Minimum lethal concentration of sodium hypochlorite for the amphibian pathogen Batrachochytrium dendrobatidis.

Sodium hypochlorite (NaOCl) is the active ingredient in household bleach and is commonly used as a disinfectant to clean equipment contaminated by the amphibian pathogen Batrachochytrium dendrobatidis (Bd) in lab husbandry and field studies. We conducted a series of replicated exposure trials using a single Global Pandemic Lineage Bd isolate from Panama (JEL 310) and concentrations of NaOCl ranging from 0.006% to 0.6% for exposure times ranging from 30 seconds to 15 minutes to determine the minimum lethal concentration of NaOCl for this isolate of Bd. Sodium hypochlorite completely killed Bd at a concentration of 0.03% during a 15-minute exposure time, while 0.12% NaOCl was effective at all exposure times (30s-15min).

Cell Density Effects of Frog Skin Bacteria on Their Capacity to Inhibit Growth of the Chytrid Fungus, Batrachochytrium dendrobatidis.

Bacterial symbionts on frog skin can reduce the growth of the chytrid fungus Batrachochytrium dendrobatidis (Bd) through production of inhibitory metabolites. Bacteria can be effective at increasing the resistance of amphibians to chytridiomycosis when added to amphibian skin, and isolates can be screened for production of metabolites that inhibit Bd growth in vitro. However, some bacteria use density-dependent mechanism such as quorum sensing to regulate metabolite production. It is therefore important to consider cell density effects when evaluating bacteria as possible candidates for bioaugmentation. The aim of our study was to evaluate how the density of cutaneous bacteria affects their inhibition of Bd growth in vitro. We sampled cutaneous bacteria isolated from three frog species in the tropical rainforests of northern Queensland, Australia, and selected ten isolates that were inhibitory to Bd in standardised pilot trials. We grew each isolate in liquid culture at a range of initial dilutions, sub-sampled each dilution at a series of times during the first 48 h of growth and measured spectrophotometric absorbance values, cell counts and Bd-inhibitory activity of cell-free supernatants at each time point. The challenge assay results clearly demonstrated that the inhibitory effects of most isolates were density dependent, with relatively low variation among isolates in the minimum cell density needed to inhibit Bd growth. We suggest the use of minimum cell densities and fast-growing candidate isolates to maximise bioaugmentation efforts.