Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Physiological impacts of temperature variability and climate warming in hellbenders (Cryptobranchus alleganiensis).

Cold-adapted hellbender salamanders that inhabit cool mountain streams are expected to fare poorly under warmer projected climate scenarios. This study investigated the physiological consequences of long-term, naturalistic temperature variation on juvenile hellbenders under simulated current and warmer (+1.6 C) climates vs. controlled steady temperatures. Mean temperature and temperature variability were both important predictors of growth as indicated by monthly body mass change (%), stress as indicated by neutrophil:lymphocyte (N:L) ratio and bacteria-killing ability of blood. Cold exposure in hellbenders was associated with weight loss, increased N:L ratios and reduced Escherichia coli killing ability of blood, and these effects were less pronounced under a warmer climate scenario. These observations suggest that cold periods may be more stressful for hellbenders than previously understood. Growth rates peaked in late spring and late fall around 14-17°C. Hellbenders experiencing warmer simulated climates retained body condition better in winter, but this was counter-balanced by a prolonged lack of growth in the 3-month summer period leading up to the fall breeding season where warmer simulated conditions resulted in an average loss of -0.6% body mass/month, compared to a gain +1.5% body mass/month under current climate scenario. Hellbenders can physiologically tolerate projected warmer temperatures and temperature fluctuations, but warmer summers may cause animals to enter the fall breeding season with a caloric deficit that may have population-level consequences.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki).

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9 GnRH; 4 µg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 µg/gbw GnRH-A + 10 µg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (<50%) as well as DNA integrity (32.8% of intact cells) after 23 min while morphology did not differ (30.4% of normal cells), regardless of the hormone stimulation. This is the first systematic study on the effect of extracellular environment over time on A. zeteki sperm quality. This will contribute to the development of sperm handling protocols and reproductive technologies for this and other endangered Atelopus species.