Non-lethal isolation of the fungal pathogen Batrachochytrium dendrobatidis (Bd) from amphibians.

The ability to isolate and purify pathogens is important for the study of infectious disease. A protocol for isolating Batrachochytrium dendrobatidis (Bd), a lethal pathogen of amphibians, has been available for over a decade, but the method relies on sacrificing infected animals. We validated a non-lethal protocol for Bd isolation that uses biopsy punches from toe webbing to collect skin samples from live amphibians in remote field locations. We successfully isolated Bd from the Cascades frog Rana cascadae and found a positive association between Bd infection and probability of Bd growth in culture. Recapture rates of sampled animals suggest that our isolation protocol did not affect frog survival. The ability to collect isolates from live animals will facilitate investigations of the biology of Bd and enhance amphibian conservation efforts.

Itraconazole treatment reduces Batrachochytrium dendrobatidis prevalence and increases overwinter field survival in juvenile Cascades frogs.

The global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to widespread extirpation of amphibian populations. During an intervention aimed at stabilizing at-risk populations, we treated wild-caught Cascades frogs Rana cascadae with the antifungal drug itraconazole. In fall 2012, we collected 60 recently metamorphosed R. cascadae from 1 of the 11 remnant populations in the Cascades Mountains (CA, USA). Of these, 30 randomly selected frogs were treated with itraconazole and the other 30 frogs served as experimental controls; all were released at the capture site. Bd prevalence was low at the time of treatment and did not differ between treated frogs and controls immediately following treatment. Following release, Bd prevalence gradually increased in controls but not in treated frogs, with noticeable (but still non-significant) differences 3 wk after treatment (27% [4/15] vs. 0% [0/13]) and strong differences 5 wk after treatment (67% [8/12] vs. 13% [1/8]). We did not detect any differences in Bd prevalence and load between experimental controls and untreated wild frogs during this time period. In spring 2013, we recaptured 7 treated frogs but none of the experimental control frogs, suggesting that over-winter survival was higher for treated frogs. The itraconazole treatment did appear to reduce growth rates: treated frogs weighed 22% less than control frogs 3 wk after treatment (0.7 vs. 0.9 g) and were 9% shorter than control frogs 5 wk after treatment (18.4 vs. 20.2 mm). However, for critically small populations, increased survival of the most at-risk life stage could prevent or delay extinction. Our results show that itraconazole treatment can be effective against Bd infection in wild amphibians, and therefore the beneficial effects on survivorship may outweigh the detrimental effects on growth.

Assessing changes in amphibian population dynamics following experimental manipulations of introduced fish.

Sport-fish introductions are now recognized as an important cause of amphibian decline, but few researchers have quantified the demographic responses of amphibians to current options in fisheries management designed to minimize effects on sensitive amphibians. Demographic analyses with mark-recapture data allow researchers to assess the relative importance of survival, local recruitment, and migration to changes in population densities. I conducted a 4-year, replicated whole-lake experiment in the Klamath Mountains of northern California (U.S.A.) to quantify changes in population density, survival, population growth rate, and recruitment of the Cascades frog (Rana cascadae) in response to manipulations of non-native fish populations. I compared responses of the frogs in lakes where fish were removed, in lakes in their naturally fish-free state, and in lakes where fish remained that were either stocked annually or no longer being stocked. Within 3 years of fish removals from 3 lakes, frog densities increased by a factor of 13.6. The survival of young adult frogs increased from 59% to 94%, and realized population growth and recruitment rates at the fish-removal lakes were more than twice as high as the rates for fish-free reference lakes and lakes that contained fish. Population growth in the fish-removal lakes was likely due to better on-site recruitment of frogs to later life stages rather than increased immigration. The effects on R. cascadae of suspending stocking were ambiguous and suggested no direct benefit to amphibians. With amphibians declining worldwide, these results show that active restoration can slow or reverse the decline of species affected by fish stocking within a short time frame.

CITIZEN SCIENTISTS MONITOR A DEADLY FUNGUS THREATENING AMPHIBIAN COMMUNITIES IN NORTHERN COASTAL CALIFORNIA, USA.

Ecoclub youth and supervising family members conducted citizen science to assess regional prevalence and distribution of Batrachochytrium dendrobatidis (Bd) among amphibians at Humboldt Bay National Wildlife Refuge (Refuge) and Redwood National and State Parks (Parks), Humboldt County, California, US, May 2013 through December 2014. Using quantitative real-time PCR, 26 (17%) of 155 samples were positive for Bd. Positive samples occurred in four frog and toad species: foothill yellow-legged frog ( Rana boylii ), northern red-legged frog ( Rana aurora ), Pacific chorus frog ( Pseudacris regilla ), and western toad (Anaxyrus [Bufo] boreas); no salamanders or anuran larvae were positive. Except for R. aurora , all infected anurans were first-time species reports for coastal northern California. At the Refuge, significantly fewer (6/71) postmetamorphic amphibians were positive compared to the Parks (20/69; P=0.0018). We assessed the association of being PCR-positive for Bd, season of sampling, and age of sampler (child, teen, or adult). The full model with season, species, and sampler age had the greatest support. Frogs tested in winter or spring were more likely to be positive than those tested in summer or fall; foothill yellow-legged frogs, northern red-legged frogs, and western toads were more likely to be positive than were Pacific chorus frogs; and the probability of being positive nearly doubled when a child (≤12 yr old) collected the sample compared to a teen or adult. Our results support other chytrid studies that found amphibians are more susceptible to Bd when temperatures are cool and that species differ in their susceptibility. The Ecoclub's findings provide new information important to conservation of northern California's coastal amphibians and demonstrate the value of involving children in citizen science.

Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila.

Diverse types of epithelial morphogenesis drive development. Similar cytoskeletal and cell adhesion machinery orchestrate these changes, but it is unclear how distinct tissue types are produced. Thus, it is important to define and compare different types of morphogenesis. We investigated cell flattening and elongation in the amnioserosa, a squamous epithelium formed at Drosophila gastrulation. Amnioserosa cells are initially columnar. Remarkably, they flatten and elongate autonomously by perpendicularly rotating the microtubule cytoskeleton--we call this 'rotary cell elongation'. Apical microtubule protrusion appears to initiate the rotation and microtubule inhibition perturbs the process. F-actin restrains and helps orient the microtubule protrusions. As amnioserosa cells elongate, they maintain their original cell-cell contacts and develop planar polarity. Myosin II localizes to anterior-posterior contacts, while the polarity protein Bazooka (PAR-3) localizes to dorsoventral contacts. Genetic analysis revealed that Myosin II and Bazooka cooperate to properly position adherens junctions. These results identify a specific cellular mechanism of squamous tissue morphogenesis and molecular interactions involved.