Rana Cascadae Research Articles

Stress and chytridiomycosis: Exogenous exposure to corticosterone does not alter amphibian susceptibility to a fungal pathogen

Recent emergence and spread of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (Bd) has been attributed to a number of factors, including environmental stressors that increase host susceptibility to Bd. Physiological stress can increase circulating levels of the hormone, corticosterone, which can alter a host's physiology and affect its susceptibility to pathogens. We experimentally elevated whole-body levels of corticosterone in both larval and post-metamorphic amphibians, and subsequently tested their susceptibility to Bd. Larvae of three species were tested (Anaxyrus boreas, Rana cascadae, and Lithobates catesbeianus) and one species was tested after metamorphosis (R. cascadae). After exposure to Bd, we measured whole-body corticosterone, infection, mortality, growth, and development. We found that exposure to exogenous corticosterone had no effect on Bd infection in any species or at either life stage. Species varied in whole-body corticosterone levels and exposure to corticosterone reduced mass in A. boreas and R. cascadae larvae. Exposure to Bd did not affect mortality, but had a number of sublethal effects. Across species, larvae exposed to Bd had higher corticosterone levels than unexposed larvae, but the opposite pattern was found in post-metamorphic R. cascadae. Bd exposure also increased larval length in all species and increased mass in R. cascadae larvae. Our results indicate that caution is warranted in assuming a strong link between elevated levels of corticosterone and disease susceptibility in amphibians. The role of physiological stress in altering Bd prevalence in amphibian populations is likely much more complicated than can be explained by examining a single "stress" endpoint.

Factors Mediating Co-Occurrence of an Economically Valuable Introduced Fish and Its Native Frog Prey

Habitat characteristics mediate predator-prey coexistence in many ecological systems but are seldom considered in species introductions. When economically important introduced predators are stocked despite known negative impacts on native species, understanding the role of refuges, landscape configurations, and community interactions can inform habitat management plans. We measured these factors in basins with introduced trout (Salmonidae) and the Cascades frog (Rana cascadae) to determine, which are responsible for observed patterns of co-occurrence of this economically important predator and its native prey. Large, vegetated shallows were strongly correlated to co-occurrence, and R. cascadae larvae occur in shallower water when fish are present, presumably to escape predation. The number of nearby breeding sites of R. cascadae was also correlated to co-occurrence, but only when the western toad (Anaxyrus boreas) was present. Because A. boreas larvae are unpalatable to fish and resemble R. cascadae, they may provide protection from trout via Batesian mimicry. Although rescue-effect dispersal from nearby populations may maintain co-occurrence, within-lake factors proved more important for predicting co-occurrence. Learning which factors allow co-occurrence between economically important introduced species and their native prey enables managers to make better-informed stocking decisions.

Temporal patterns in immunity, infection load and disease susceptibility: understanding the drivers of host responses in the amphibian‐chytrid fungus system

Summary Many pathogens infect a wide range of host species. However, variation in the outcome of infection often exists amongst hosts and is shaped by intrinsic host traits. For example, contact with pathogens may trigger changes in hosts directed toward preventing, fighting, or tolerating infection. Host responses to infection are dynamic; they change over time and vary depending on health, condition and within the context of the environment. Immunological defences are an important class of responses that mediate host–pathogen dynamics. Here, we examined temporal patterns of immunity in two amphibian species, Pacific tree frogs (Pseudacris regilla) and Cascades frogs (Rana cascadae), exposed to control conditions or experimental inoculation with the emerging infectious fungal pathogen, Batrachochytrium dendrobatidis (Bd). For each species, we compared bacterial killing ability of blood and differential white blood cell counts at four different time‐points after pathogen inoculation. We also quantified infection load over time and monitored survival. We detected qualitative and quantitative differences in species responses to Bd. Pseudacris regilla exhibited an increase in infection load over time and 16% of Bd‐exposed animals died during the experiment. Tree frogs lacked robust treatment differences in immune responses, but Bd‐exposed P. regilla tended to display weaker bacterial killing responses than unexposed control animals. Neutrophil counts did not vary consistently with treatment and lymphocytes tended to be less abundant in Bd‐exposed animals at the later sampling time‐points. In contrast, Bd‐exposed R. cascadae exhibited a decrease in infection load over time and no mortality occurred in the Bd treatment. Bd‐exposed Cascades frogs showed stronger bacterial killing responses and an elevated number of neutrophils in blood when compared with control animals, and both responses were upregulated within 48 h of pathogen exposure. Lymphocyte counts did not vary significantly with treatment. Although only statistically significant in Cascades frogs, neutrophil:lymphocyte ratios showed a trend of being elevated in Bd‐exposed animals of both species and are indicative of pathogen‐induced physiological stress. Our results suggest that variation in systemic immunological responses of two syntopic amphibian species is associated with and may contribute to differential patterns of survival and infection load during exposure to the chytrid fungus. Species variation in immunological responses as soon as 48 h after pathogen exposure suggests that initial host–pathogen interactions may set the stage for subsequent infection and disease progression. Variation in host responses can drive disease dynamics and comparative studies of host responses to pathogens are critical for making predictions about pathogen emergence, spread and persistence.

The amphibian skin‐associated microbiome across species, space and life history stages

Skin-associated bacteria of amphibians are increasingly recognized for their role in defence against pathogens, yet we have little understanding of their basic ecology. Here, we use high-throughput 16S rRNA gene sequencing to examine the host and environmental influences on the skin microbiota of the cohabiting amphibian species Anaxyrus boreas, Pseudacris regilla, Taricha torosa and Lithobates catesbeianus from the Central Valley in California. We also studied populations of Rana cascadae over a large geographic range in the Klamath Mountain range of Northern California, and across developmental stages within a single site. Dominant bacterial phylotypes on amphibian skin included taxa from Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria, Firmicutes, Sphingobacteria and Actinobacteria. Amphibian species identity was the strongest predictor of microbial community composition. Secondarily, within a given amphibian species, wetland site explained significant variation. Amphibian-associated microbiota differed systematically from microbial assemblages in their environments. Rana cascadae tadpoles have skin bacterial communities distinct from postmetamorphic conspecifics, indicating a strong developmental shift in the skin microbes following metamorphosis. Establishing patterns observed in the skin microbiota of wild amphibians and environmental factors that underlie them is necessary to understand skin symbiont community assembly, and ultimately, the role skin microbiota play in the extended host phenotype including disease resistance.

Variations in lethal and sublethal effects of cypermethrin among aquatic stages and species of anuran amphibians

Despite the use of model species to predict the effects of chemicals in the environment, unpredicted variation in levels of risk to organisms from xenobiotics can be observed. Physiological and morphological differences between species and life stages may lead to differences in sensitivity, while seasonal and spatial variation in pesticide concentrations may affect the level of risk faced by organisms in the environment. Because anurans breed in aquatic habitats subject to contamination by runoff and spraying, they are particularly vulnerable to pesticides. In the present study, embryos, newly hatched larvae, and larvae with limb buds of 3 anuran amphibian species--Pseudacris regilla, Rana cascadae, and Rana aurora--were exposed for 48 h to either 0.5 µg/L or 5.0 µg/L cypermethrin under laboratory conditions. The authors monitored hatching success, larval survival, and measured growth. Additionally, they assayed avoidance behavior 2 wk after exposure or 2 wk after hatching for individuals exposed as embryos. Hatching and survival were not affected in animals of any species exposed as embryos. After exposure as embryos and as newly hatched larvae, however, P. regilla displayed behavioral abnormalities in response to prodding. Cypermethrin increased mortality in P. regilla exposed in both larval stages. Cypermethrin also increased mortality in larval R. cascadae when exposed at the early stage. These results indicate variation in sensitivity to environmentally relevant concentrations of cypermethrin among anuran species and life stages.

Development and infectious disease in hosts with complex life cycles.

Metamorphosis is often characterized by profound changes in morphology and physiology that can affect the dynamics of species interactions. For example, the interaction between a pathogen and its host may differ depending on the life stage of the host or pathogen. One pathogen that infects hosts with complex life cycles is the emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd). We sought to determine how conditions at the larval stage can affect variation in development and patterns of Bd infection across amphibian life stages. We used outdoor experimental mesocosms to simulate natural pond habitats and manipulated the presence of Bd, the larval density, and the number of host species in larvae of two co-occurring amphibian species (Rana cascadae and Pseudacris regilla). We found that infection differed between species throughout development; P. regilla consistently had higher infection severity compared to R. cascadae. Additionally, while up to 100% of larvae were infected, only 18.2% of R. cascadae and 81.5% of P. regilla were infected after metamorphosis. This indicates that amphibians have the ability to recover from Bd infection as they undergo metamorphosis. Higher larval densities in P. regilla led to a shorter larval period, and individuals with a shorter larval period had lower infection severity. This led to a trend where P. regilla larvae reared at high densities tended to have lower infection prevalence after metamorphosis. We also found that exposure to Bd increased larval mortality and prolonged the larval period in P. regilla, indicating that P. regilla are susceptible to the negative effects of Bd as larvae. This study demonstrates that host density, species composition, and pathogen exposure may all interact to influence development and infection in hosts with complex life cycles.

Host identity matters in the amphibian-Batrachochytrium dendrobatidis system: fine-scale patterns of variation in responses to a multi-host pathogen.

Species composition within ecological assemblages can drive disease dynamics including pathogen invasion, spread, and persistence. In multi-host pathogen systems, interspecific variation in responses to infection creates important context dependency when predicting the outcome of disease. Here, we examine the responses of three sympatric host species to a single fungal pathogen, Batrachochytrium dendrobatidis, which is associated with worldwide amphibian population declines and extinctions. Using an experimental approach, we show that amphibian species from three different genera display significant differences in patterns of pathgen-induced mortality as well as the magnitude and temporal dynamics of infection load. We exposed amphibians to one of four inoculation dose treatments at both larval and post- metamorphic stages and quantified infection load on day 8 and day 15 post-inoculation. Of the three species examined, only one (the Pacific treefrog; Pseudacris regilla) displayed “dose-dependent” responses; survival was reduced and infection load was elevated as inoculation dose was increased. We observed a reduction in survival but no differences in infection load across pathogen treatments in Cascades frogs (Rana cascadae). Western toads (Anaxyrus boreas) displayed differences in infection load but no differences in survival across pathogen treatments. Within species, responses to the pathogen varied with life history stage, and the most heavily infected species at the larval stage was different from the most heavily infected species at the post-metamorphic stage. Temporal changes in infection load were species and life history stage-specific. We show that variation in susceptibility to this multi-host pathogen is complex when viewed at a fine-scale and may be mediated through intrinsic host traits.

Contaminant residues and declines of the Cascades frog (Rana cascadae) in the California Cascades, USA

Populations of Cascades frogs (Rana cascadae) have declined precipitously in the Mount Lassen area, but remain abundant in the other half of their California range in the Klamath Mountains. To evaluate the role of contaminants in Cascade frog declines, we sampled sediment and frog tadpole tissue at 31 sites where Cascades frogs had disappeared and sites where Cascades frogs are still present across the Lassen and Klamath regions. Pacific chorus frogs (Pseudacris regilla) were tested and used as surrogates for residue concentrations in Cascades frogs. We analyzed a total of 79 tadpole samples for 73 semivolatile contaminants including pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). The most frequently detected residue was endosulfan sulfate, followed by dacthal, chlorpyrifos, PCB 187, endosulfan II, trans-chlordane, and trans-nonachlor. Chorus frogs had similar residue concentrations as Cascades frogs for most but not all chemicals, indicating that chorus frogs can serve as a reasonable proxy for chemical concentrations in Cascades frogs. None of the contaminants in tissue or sediment had significantly higher concentrations at sites where Cascades frogs have disappeared than at sites where Cascades frogs are still present. We found no evidence to support the hypothesis that the contaminants analyzed have contributed to the decline of Cascades frogs in northern California, although we were able to analyze only a handful of the over 300 pesticides currently used in the area.

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.

The effects of multiple stressors on wetland communities: pesticides, pathogens and competing amphibians

Summary1. Anthropogenic effects have propelled us into what many have described as the sixth mass extinction, and amphibians are among the most affected groups. The causes of global amphibian population declines and extinctions are varied, complex and context‐dependent and may involve multiple stressors. However, experimental studies examining multiple factors contributing to amphibian population declines are rare.2. Using outdoor mesocosms containing zooplankton, phytoplankton, periphyton and tadpoles, we conducted a 2 × 2 × 3 factorial experiment that examined the separate and combined effects of an insecticide and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on three different assemblages of larval pacific treefrogs (Pseudacris regilla) and Cascades frogs (Rana cascadae).3. Larval amphibian growth and development were affected by carbaryl and the amphibian assemblage treatment, but only minimally by Bd. Carbaryl delayed metamorphosis in both amphibian species and increased the growth rate of P. regilla. Carbaryl also reduced cladoceran abundance, which, in turn, had positive effects on phytoplankton abundance but no effect on periphyton biomass. Substituting 20 intraspecific competitors with 20 interspecific competitors decreased the larval period but not the growth rate of P. regilla. In contrast, substituting 20 intraspecific competitors with 20 interspecific competitors had no effect on R. cascadae. Results of real‐time quantitative polymerase chain reaction (qPCR) analysis confirmed infection of Bd‐exposed animals, but exposure to Bd had no effects on either species in univariate analyses, although it had significant or nearly significant effects in several multivariate analyses. In short, we found no interactive effects among the treatments on amphibian growth and development.4. We encourage future research on the interactive effects of pesticides and pathogens on amphibian communities.

Factors related to the distribution and prevalence of the fungal pathogen Batrachochytrium dendrobatidis in Rana cascadae and other amphibians in the Klamath Mountains

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been associated with declines and extinctions of montane amphibians worldwide. To gain insight into factors affecting its distribution and prevalence we focus on the amphibian community of the Klamath Mountains in northwest California. The Cascades frog (Rana cascadae), one of the most common amphibians in these mountains, experienced increased mortality as a result of Bd exposure in laboratory trials and has experienced recent, dramatic declines in other parts of California. We surveyed 112 sites in the Klamaths, all of which supported R. cascadae between 1999 and 2002, for amphibians and Bd to (1) determine the distribution of Bd, (2) evaluate changes in the distribution of R. cascadae, and (3) assess associations between potential biotic and abiotic drivers and Bd infection. Bd was widely distributed in the Klamath Mountains – we detected the pathogen at 64% of sites. R. cascadae was found at 79% of sites, and was often infected with Bd. These results suggest that Bd has not caused dramatic declines in R. cascadae in the Klamaths in recent years. Subadult R. cascadae had a higher Bd prevalence than other R. cascadae life stages (subadults: 36%, adults: 25%, metamorphs: 4%, larvae: 1%), and while the probability of infection decreased over the season for adults, it did not for subadults, suggesting that subadults may be more vulnerable to chytridiomycosis than other R. cascadae life stages. Bd prevalence in R. cascadae was highest early in the season at high-elevation sites, which may indicate that populations inhabiting high elevation sites may have a greater risk of being affected by chytridiomycosis. Three other common amphibian species also tested positive for Bd: Pacific chorus frog (Pseudacris regilla), western toad (Anaxyrus boreas), and rough-skinned newt (Taricha granulosa).

Toxic Effects of the Herbicide Roundup® Regular on Pacific Northwestern Amphibians

One of the most widely used herbicides for commercial and home use is glyphosate, the active ingredient in Roundup® Regular. We examined toxicity of the herbicide Roundup® on 6 amphibian species: Ambystoma gracile, Ambystoma macrodactylum, Anaxyrus [Bufo] boreas, Pseudacris regilla, Rana cascadae, and Rana luteiventris. Larvae were exposed to 6 different Roundup® Regular treatments (0 (control), 0.1, 0.5, 1.0, 2.0, and 5.0 mg AI/L dilutions of glyphosate) and monitored for 16 d. Estimated acute lethal concentrations at 24 h (LC50) varied significantly among species (ANOVA, F(3, 56) = 3.54, p < 0.0202), with concentrations ranging from 0.43 mg AI/L of Roundup® for P. regilla to 2.66 mg AI/L for A. boreas. Bufonid and ambystomatid larvae were less sensitive than Ranid and Pseudacrid species tested, with no salamander larval mortality occurring at 24 h. Mean time-to-death varied from 1 d for P. regilla to 8.3 d for A. gracile, respectively (ANOVA, F(5, 971) = 108, p < 0.0001). For exposure times longer than 24 h, the A. boreas was not significantly different than the salamanders for time-to-death, based on Tukey-Kramer comparisons. Results suggest Roundup® Regular is highly toxic to the amphibians at levels below EPA standards for drinking water and at concentrations they may be exposed to during overspray. We recommend the use of less toxic glyphosate-based herbicides in aquatic systems, if applications are necessary, or made during times of year when amphibian larvae are not present.

Indirect effects of introduced trout on Cascades frogs (Rana cascadae) via shared aquatic prey

Summary 1. The introduction of trout to montane lakes has negatively affected amphibian populations across the western United States. In northern California’s Klamath–Siskiyou Mountains, introduced trout have diminished the distribution and abundance of a native ranid frog, Rana (=Lithobates) cascadae. This is primarily thought to be the result of predation on frog larvae. However, if trout feed on larval aquatic insects that are available to R. cascadae only after emergence, then resource competition may also affect this declining native amphibian. 2. Stomach contents of R. cascadae were compared between lakes that contained trout and those from which introduced trout were removed. Total prey mass in stomach contents relative to frog body mass was not significantly different between lakes with fish and fish-removal lakes, but in the former R. cascadae consumed a smaller proportion of adult aquatic insects. The stomach contents of fish included larvae of aquatic insects that are, as adults, eaten by R. cascadae. 3. Rana cascadae consumed fewer caddisflies (Trichoptera) and more grasshoppers (Orthoptera) at lakes with higher densities of fish. At lakes with greater aquatic habitat complexity, R. cascadae consumed more water striders (Hemiptera: Gerridae) and terrestrial spiders (Araneae). 4. We suggest that reductions in the availability of emerging aquatic insects cause R. cascadae to consume more terrestrial prey where trout are present. Thus, introduced trout may influence native amphibians directly through predation and, indirectly, through pre-emptive resource competition.

Water clarity, maternal behavior, and physiology combine to eliminate UV radiation risk to amphibians in a montane landscape

Increasing UV-B radiation (UV-B; 290-320 nm) due to stratospheric ozone depletion has been a leading explanation for the decline in amphibians for nearly 2 decades. Yet, the likelihood that UV-B can influence amphibians at the large spatial scales relevant to population declines has not yet been evaluated. A key limitation has been in relating results from individual sites to the effect of UV-B for populations distributed across heterogeneous landscapes. We measured critical embryonic exposures to UV-B for two species of montane amphibians with contrasting physiological sensitivities, long-toed salamander (Ambystoma macrodactylum) and Cascades frog (Rana cascadae), at field sites spanning a gradient of UV-B attenuation in water. We then used these experimental results to estimate the proportion of embryos exposed to harmful UV-B across a large number of breeding sites. By combining surveys of the incubation timing, incident UV-B, optical transparency of water, and oviposition depth and light exposure of embryos at each site, we present a comprehensive assessment of the risk posed by UV-B for montane amphibians of the Pacific Northwest. We found that only 1.1% of A. macrodactylum and no R. cascadae embryos across a landscape of breeding sites are exposed to UV-B exceeding lethal levels. These results emphasize that accurately estimating the risk posed by environmental stressors requires placing experimental results in a broader ecological context that accounts for the heterogeneity experienced by populations distributed across natural landscapes.

Predatory Leeches (Hirudinida) May Contribute to Amphibian Declines in the Lassen Region, California

Researchers have documented precipitous declines in Cascades Frog (Rana cascadae) populations in the southern portion of the species' range, in the Lassen region of California. Reasons for the declines, however, have not been elucidated. In addition to common, widespread causes, an understanding of local community interactions may be necessary to fully understand proximal causes of the declines. Based on existing literature and observations made during extensive aquatic surveys throughout the range of R. cascadae in California, we propose that a proliferation of freshwater leeches (subclass Hirudinida) in the Lassen region may be adversely affecting R. cascadae populations. Leeches may affect R. cascadae survival or fecundity directly by preying on egg and hatchling life stages, and indirectly by contributing to the spread of pathogens and secondary parasites. In 2007, we conducted focused surveys at known or historic R. cascadae breeding sites to document co-occurrences of R. cascadae and leeches, determine if leeches were preying on or parasitizing eggs or hatchlings of R. cascadae, and identify the leech species to establish whether or not they were native to the region. We found R. cascade at 4 of 21 sites surveyed and freshwater leeches at 9 sites, including all sites with R. cascadae. In 2007 and 2008, the predatory leech Haemopis marmorata frequented R. cascadae egg masses, was observed probing or tearing at eggs on 24 occasions, and was 10 times more common in 1-m2 plots centered on egg masses than in similar plots without egg masses. Six species of leech were identified from the Lassen region, only 3 of which have been documented from the region prior to this study. Given our documentation of a diverse freshwater leech fauna in direct association with a precipitously declining species, we believe a better understanding of the biology and ecology of this poorly studied taxon is needed, in addition to studies of the effects of freshwater leeches on survival and recruitment of co-occurring amphibians.

Sublethal predators and their injured prey: linking aquatic predators and severe limb abnormalities in amphibians

While many predators completely consume their prey, others feed only on blood or tissue without killing the prey, sometimes causing ecologically significant levels of injury. We investigated the importance of sublethal predator attacks in driving an emerging issue of conservation importance: missing-limb deformities in amphibians. We combined long-term field data and manipulative experiments to evaluate the role of sublethal predation in causing abnormalities in two regions of central Oregon, U.S.A. Since 1988, western toads (Bufo boreas) in Lake Aspen have exhibited abnormalities dominated by partially missing limbs and digits at annual frequencies from <1% to 35%. On Broken Top volcano, we found comparable types and frequencies of abnormalities in Cascades frogs (Rana cascadae). Field sampling and observational data implicated two aquatic predators in these abnormality phenomena: introduced sticklebacks (Gasterosteus aculeatus) at Lake Aspen and corduliid dragonfly larvae (Somatochlora albicincta) at Broken Top. In experiments, these predators produced limb abnormalities identical to those observed in the respective regions. At Lake Aspen, in situ predator exclosures effectively eliminated abnormalities in toads, while comparisons among years with low and high stickleback abundance and between wetlands with and without sticklebacks reinforced the link between fish and amphibian abnormalities. Neither trematode parasite infection nor pesticide contamination could explain observed abnormalities. Our results suggest that predators are an important explanation for missing-limb abnormalities and highlight the ecological significance of sublethal predation in nature.

Evaluating Amphibian Declines with Site Revisits and Occupancy Models: Status of Montane Anurans in the Pacific Northwest USA

Amphibian declines have been reported in mountainous areas around the western USA. Few data quantify the extent of population losses in the Pacific Northwest, a region in which amphibian declines have received much attention. From 2001–2004, we resurveyed historical breeding sites of two species of conservation concern, the Western Toad (Bufo [=Anaxyrus] boreas) and Cascades Frog (Rana cascadae). We detected B. boreas breeding at 75.9% and R. cascadae breeding at 66.6% of historical sites. When we analyzed the data using occupancy models that accounted for detection probability, we estimated the current use of historically occupied sites in our study area was 84.9% (SE = 4.9) for B. boreas and 72.4% (SE = 6.6) for R. cascadae. Our ability to detect B. boreas at sites where they were present was lower in the first year of surveys (a low snowpack year) and higher at sites with introduced fish. Our ability to detect R. cascadae was lower at sites with fish. The probability that B. boreas still uses a historical site for breeding was related to the easting of the site (+) and the age of record (-). None of the variables we analyzed was strongly related to R. cascadae occupancy. Both species had increased odds of occupancy with higher latitude, but model support for this variable was modest. Our analysis suggests that while local losses are possible, these two amphibians have not experienced recent, broad population losses in the Oregon Cascades. Historical site revisitation studies such as ours cannot distinguish between population losses and site switching, and do not account for colonization of new habitats, so our analysis may overestimate declines in occupancy within our study area.

Amphibian Chytridiomycosis in the Oregon Spotted Frog (RANA PRETIOSA) IN WASHINGTON STATE, USA

In recent years Batrachochytrium dendrobatidis (Bd), a chytridiomycete fungus pathogenic to amphibians (Longcore and others 1999), has been implicated as the proximate cause of amphibian declines around the world (Berger and others 1998; Daszak and others 2003; Muths and others 2003; Pounds and others 2006). Despite the insidious nature of Bd (Green and others 2002), few published data exist addressing its occurrence in the Pacific Northwest (PNW). When Pearl and others (2007) opportunistically examined 7 PNW amphibian species, they found Bd most often (57% of 21 individuals from 14 sampled populations) in the highly aquatic Oregon Spotted Frog (Rana pretiosa), an Endangered Species in Washington State (WDFW 2009). However, all R. pretiosa they sampled were from Oregon, and Bd was not detected in the Rana cascadae (Cascades Frog) and Anaxyrus boreas (Western Toad) specimens they sampled from Washington. Here, we report the detection of Bd in R. pretiosa from Washington. Since 1997 and 1998, respectively, we have been monitoring R. pretiosa populations at the Trout Lake Natural Area Preserve (TLNAP: UTM Zone 10, 610857-612950E, 5095880-5097574N, WGS84; elev. 594 to 599 m) and Conboy Lake National Wildlife Refuge (CLNWR: UTM Zone 10, 625223-635180E, 5086652-5095491N, WGS84; elev. 552 to 576 m), in Klickitat County, Washington. These sites represent 2 of only 3 areas where R. pretiosa is known to occur in Washington (McAllister and Leonard 1997). At both sites, monitoring included egg mass surveys during the annual breeding season following snowmelt (late February to mid-March at CLNWR; midMarch to early April at TLNAP). Surveys involved area-specific counts of individual egg masses and egg mass groups, each of which was marked with flags and geo-referenced using a Geographic Positioning System (GPS) to avoid double-counting. Sampling at CLNWR addressed 4 hydrologically distinct units, which were surveyed repeatedly until no new egg masses were found; at TLNAP, 3 units consistently used for breeding were sampled. We inferred a 1:1 correspondence between egg mass numbers and number of breeding females based on a combination of our direct observations of oviposition (n 5 13), the recapture of non-gravid females known to have laid eggs (n 5 84), and the relatively short interval over which the laying of new egg masses occurs (about 3 wk). Collectively, these data indicate that females lay only 1 clutch annually, and that egg mass numbers reflect the effective population numbers of adult females. Decline in egg mass numbers in 3 of the 4 surveyed units at CLNWR from 2004 to 2005 (Fig. 1) and 2 of the 3 units at TLNAP over the same period (Fig. 2), coupled with the increasing recognition of Bd as a cause of amphibian declines, motivated us to collect dead frogs found during 2006 and test them for Bd. Five dead adult R. pretiosa were tested for Bd; 1 from CLNWR and 4 from TLNAP. The CLNWR specimen collected on 13 March 2006 displayed feeble vital signs, minimal response to touch, and righting response was lacking. The frog died within 20 min of discovery, was preserved in 10% formalin, and then stored in 70% ethanol before histological examination using a standard wet-mount preparation of its epidermal tissue (Berger and others 1999), which was sloughing extensively as multiple epidermal layers. A dead adult female R. GENERAL NOTES

Experimental examination of the effects of ultraviolet-B radiation in combination with other stressors on frog larvae

Ultraviolet-B radiation (UVB) is a ubiquitous stressor with negative effects on many aquatic organisms. In amphibians, ambient levels of UVB can result in impaired growth, slowed development, malformations, altered behavior and mortality. UVB can also interact with other environmental stressors to amplify these negative effects on individuals. In outdoor mesocosm and laboratory experiments we studied potential synergistic effects of UVB, a pathogenic fungus, Batrachochytrium dendrobatidis (Bd), and varying temperatures on larval Cascades frogs (Rana cascadae). First, we compared survivorship, growth and development in two mesocosm experiments with UVB- and Bd-exposure treatments. We then investigated the effects of UVB on larvae in the laboratory under two temperature regimes, monitoring survival and behavior. We found reduced survival of R. cascadae larvae with exposure to UVB radiation in all experiments. In the mesocosm experiments, growth and development were not affected in either treatment, and no effect of Bd was found. In the laboratory experiment, larvae exposed to UVB demonstrated decreased activity levels. We also found a trend towards reduced survival when UVB and cold temperatures were combined. Our results show that amphibian larvae can suffer both lethal and sublethal effects when exposed to UVB radiation.

Effects of the pathogenic water mold Saprolegnia ferax on survival of amphibian larvae

Infectious diseases are a significant threat to worldwide biodiversity. Amphibian declines, a significant part of current biodiversity losses, are in many cases associated with infectious disease. Water molds are one group of pathogens affecting amphibians on a worldwide basis. Although water molds have been studied extensively for their effects on host embryos, little information is available about how they affect post-embryonic amphibians. We tested the effects of one species of water mold, Saprolegnia ferax, in a comparative study of larvae of 4 amphibian species: Pseudacris regilla (Pacific treefrog), Rana cascadae (Cascades frog), Ambystoma macrodactylum (long-toed salamander), and R. aurora (red-legged frog). S. ferax can kill amphibians at the embryonic and juvenile life history stages, depending on the amphibian species. In the present study, a 1 wk exposure to S. ferax killed P. regilla larvae and a 2 wk exposure killed R. aurora larvae. Larvae of the other host species were unaffected after 1 wk of exposure to S. ferax. Our results suggest that S. ferax can kill amphibian larvae and further suggest that evaluation of how pathogens affect amphibians at the population level requires investigation at various life stages.