Differential host susceptibility to Batrachochytrium dendrobatidis, an emerging amphibian pathogen.

The amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) has received considerable attention due to its role in amphibian population declines worldwide. Although many amphibian species appear to be affected by Bd, there is little information on species-specific differences in susceptibility to this pathogen. We used a comparative experimental approach to examine Bd susceptibility in 6 amphibian species from the United States. We exposed postmetamorphic animals to Bd for 30 days and monitored mortality, feeding rates, and infection levels. In all species tested, Bd-exposed animals had higher rates of mortality than unexposed (control) animals. However, we found differences in mortality rates among species even though the amount of Bd detected on the different species' bodies did not differ. Of the species tested, southern toads (Anaxyrus terrestris) and wood frogs (Lithobates sylvaticus) had the highest rates of Bd-related mortality. Within species, we detected lower levels of Bd on individuals that survived longer and found that the relationship between body size and infection levels differed among species. Our results indicate that, even under identical conditions, amphibian species differ in susceptibility to Bd. This study represents a step toward identifying and understanding species variation in disease susceptibility, which can be used to optimize conservation strategies.

Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis.

When exploring the possible factors contributing to population declines, it is necessary to consider multiple, interacting environmental stressors. Here, we investigate the impact of 2 factors, ultraviolet radiation and disease, on the survival of anuran amphibians. Exposure to ultraviolet-B (UV-B) radiation increases mortality and results in various sub-lethal effects for many amphibian species. Infectious diseases can also negatively impact amphibian populations. In this study, we exposed metamorphic individuals (metamorphs) to both UV-B and Batrachochytrium dendrobatidis (BD), a fungal pathogen and cause of the disease chytridiomycosis, and monitored survival for 3 wk. We tested for possible interactions between UV-B and BD in 3 species: the Cascades frog Rana cascadae; the Western toad Bufo boreas; and the Pacific treefrog Hyla regilla. We found strong interspecific differences in susceptibility to BD. For example, R. cascadae suffered a large increase in mortality when exposed to BD; B. boreas also experienced mortality, but this effect was small relative to the R. cascadae response. H. regilla did not show any decrease in survival when exposed to either factor. No synergistic interactions between UV-B and BD were found for any of the test species. A previous study investigating the impact of BD on larval amphibians showed different species responses (Blaustein et al. 2005a). Our results highlight the importance of studying multiple life history stages when determining the impact of environmental stressors. The contrast between these 2 studies emphasizes how vulnerability to a pathogen can vary between life history stages within a single species.

Juvenile amphibians do not avoid potentially lethal levels of urea on soil substrate.

We examined the effects of a forest fertilizer (urea) on newly metamorphosed terrestrial amphibians (Western toads, Bufo boreas; Cascades frogs, Rana cascadae; long-toed salamanders, Ambystoma macrodactylum; and roughskin newts, Taricha granulosa). We examined avoidance behavior of Western toads and Cascades frogs on both paper towel and soil substrates dosed with urea (control and 100 kg N/ha and an additional treatment of 50 kg N/ha for Western toads on soil substrate) and avoidance behavior of long-toed salamanders on soil substrate dosed with urea. We further examined the survival and feeding behavior of all four species exposed to urea on soil substrate (100 kg N/ha) for 5 d. Juvenile Western toads and Cascades frogs avoided paper towels dosed with urea but did not avoid urea-dosed soil substrate. However, Western toads and Cascades frogs both suffered significant mortality when exposed to urea on a soil substrate for 5 d. Furthermore, after adjusting for weight, we found that urea-exposed juvenile Western toads and Cascades frogs consumed significantly fewer prey items (crickets) compared with nonexposed control animals. Long-toed salamanders did not discriminate against soil substrate dosed with urea, and neither long-toed salamanders nor roughskin newts died or reduced prey consumption as a result of urea exposure. Juvenile amphibians may not be able to detect and avoid harmful levels of urea fertilizer on a natural substrate. Furthermore, anthropogenic stressors such as urea fertilizer can significantly reduce the survival and prey consumption of juvenile amphibians. These effects are important to consider in light of possible threats to the conservation status of many amphibian species.

Avoidance response of juvenile Pacific treefrogs to chemical cues of introduced predatory bullfrogs.

Bullfrogs (Rana catesbeiana), native to eastern North America, were introduced into Oregon in the 1930's. Bullfrogs are highly efficient predators that are known to eat a variety of prey including other amphibians. In laboratory experiments, we investigated whether juvenile Pacific treefrogs (Hyla regilla) recognize adult bullfrogs as a predatory threat. The ability of prey animals to acquire recognition of an introduced predator has important implications for survival of the prey. We found that treefrogs from a population that co-occurred with bullfrogs showed a strong avoidance of chemical cues of bullfrogs. In contrast, treefrogs from a population that did not co-occur with bullfrogs, did not respond to the bullfrog cues. Additional experiments showed that both populations of treefrogs use chemical cues to mediate predation risk. Treefrogs from both populations avoided chemical alarm cues from injured conspecifics.

Sensitivity to urea fertilization in three amphibian species.

Forest fertilization with granular urea is a well-established management practice in many forested regions of the world. We hypothesize that chemical forest fertilizers may be affecting forest-dwelling wildlife. In the laboratory, we studied the effects of fertilization doses of granular urea on three species of forest-dwelling amphibians (Plethodon vehiculum, Rhyacotriton variegatus, and Taricha granulosa). In avoidance experiments, the three species avoided a substrate treated with a dose of 225 kg N/ha urea. In toxicity experiments, we exposed amphibians to urea at doses of 225 kg N/ha and 450 kg N/ha for 4 days. The observed effects increased with time and dose, and there were significant differences in sensitivity among the species. Both treatment levels had an acute effect on survival of P. vehiculum and R. variegatus. At 24 h, mortality at the highest dose was 67% for P. vehiculum, and 47% for R. variegatus. In contrast, there was no mortality for T. granulosa at these concentrations. We suggest that environmental levels of urea could be affecting behavior and survival of some amphibians species in fertilized forests.

Complex causes of amphibian population declines.

Amphibian populations have suffered widespread declines and extinctions in recent decades. Although climatic changes, increased exposure to ultraviolet-B (UV-B) radiation and increased prevalence of disease have all been implicated at particular localities, the importance of global environmental change remains unclear. Here we report that pathogen outbreaks in amphibian populations in the western USA are linked to climate-induced changes in UV-B exposure. Using long-term observational data and a field experiment, we examine patterns among interannual variability in precipitation, UV-B exposure and infection by a pathogenic oomycete, Saprolegnia ferax. Our findings indicate that climate-induced reductions in water depth at oviposition sites have caused high mortality of embryos by increasing their exposure to UV-B radiation and, consequently, their vulnerability to infection. Precipitation, and thus water depth/UV-B exposure, is strongly linked to El Niño/Southern Oscillation cycles, underscoring the role of large-scale climatic patterns involving the tropical Pacific. Elevated sea-surface temperatures in this region since the mid-1970s, which have affected the climate over much of the world, could be the precursor for pathogen-mediated amphibian declines in many regions.

Combined effects of UV-B, nitrate, and low pH reduce the survival and activity level of larval cascades frogs (Rana cascadae).

We investigated interactions between low pH, high nitrate level, and ultraviolet-B (UV-B) light on the survival and activity level of larval Cascades frogs (Rana cascadae). We used a fully factorial experimental design, with pH levels of 5 and 7; initial "pulse" nitrate exposure levels of 0, 5, and 20 mg/L; and UV-B present or absent. After a 3-week laboratory exposure, we measured survival and activity level of the larvae. The experiment was repeated two times, in two separate years. Similar effects on survival and activity level were observed in both experiments. R. cascadae survival was not significantly reduced in treatments with individual factors alone (i.e., UV-B control, pH 5 control, or high nitrate level without pH or UV-B). However, in experiments from both years, survival and activity levels of larval R. cascadae were significantly reduced in the treatment with low pH, high nitrate, and UV-B together. In both years, analysis of variance (ANOVA) indicated that pH and nitrate had the greatest effect on survival and that UV-B and nitrate had the greatest effect on activity level. Additional effects were observed in the 1998 experiment on both survival and activity level. In 1998, UV radiation and the interaction term between pH and nitrate (pH x nitrate) had a significant effect on survival. Also in the 1998 experiment, activity level was significantly reduced in treatments at neutral pH with UV, at initial nitrate doses of 5 and 20 mg/L and at neutral pH without UV at an initial nitrate dose of 20 mg/L. We suggest that the adverse effects were due to the multiple stressors acting together.

Ambient UV-B radiation causes deformities in amphibian embryos.

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual's subsequent survival after hatching.

Developmental responses of amphibians to solar and artificial UVB sources: a comparative study.

Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species-Hyla regilla, Rana cascadae, and Bufo boreas-for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.

Avoidance response of a terrestrial salamander (Ambystoma macrodactylum) to chemical alarm cues.

Organisms from a wide variety of taxonomic groups possess chemical alarm cues that are important in mediating predator avoidance. However, little is known about the presence of such alarm cues in most amphibians, and in particular terrestrial salamanders. In this study we tested whether adult long-toed salamanders (Ambystoma macrodactylum) showed an avoidance response to stimuli from injured conspecifics. Avoidance of stimuli from injured conspecifics could represent avoidance of a chemical alarm cue or, alternatively, avoidance of a territorial pheromone or conspecific predator odor. Consequently, we also tested whether salamanders avoided stimuli from noninjured conspecifics. Salamanders avoided stimuli from injured but not from noninjured conspecifics. Therefore, we concluded that the response to injured conspecifics represents avoidance of a chemical alarm cue and not avoidance of a territorial pheromone or predator cue. This is the first clear demonstration of chemical alarm signaling by a terrestrial amphibian and the first report of chemical alarm signaling in an ambystomatid salamander. By avoiding an area containing stimuli from injured conspecifics, long-toed salamanders may lower their risk of predation by avoiding areas where predators are foraging.

Synergism between UV-B radiation and a pathogen magnifies amphibian embryo mortality in nature.

Previous research has shown that amphibians have differential sensitivity to ultraviolet-B (UV-B) radiation. In some species, ambient levels of UV-B radiation cause embryonic mortality in nature. The detrimental effects of UV-B alone or with other agents may ultimately affect amphibians at the population level. Here, we experimentally demonstrate a synergistic effect between UV-B radiation and a pathogenic fungus in the field that increases the mortality of amphibian embryos compared with either factor alone. Studies investigating single factors for causes of amphibian egg mortality or population declines may not reveal the complex factors involved in declines.

UV repair and resistance to solar UV-B in amphibian eggs: a link to population declines?

The populations of many amphibian species, in widely scattered habitats, appear to be in severe decline; other amphibians show no such declines. There is no known single cause for the declines, but their widespread distribution suggests involvement of global agents--increased UV-B radiation, for example. We addressed the hypothesis that differential sensitivity among species to UV radiation contributes to these population declines. We focused on species-specific differences in the abilities of eggs to repair UV radiation damage to DNA and differential hatching success of embryos exposed to solar radiation at natural oviposition sites. Quantitative comparisons of activities of a key UV-damage-specific repair enzyme, photolyase, among oocytes and eggs from 10 amphibian species were reproducibly characteristic for a given species but varied > 80-fold among the species. Levels of photolyase generally correlated with expected exposure of eggs to sunlight. Among the frog and toad species studied, the highest activity was shown by the Pacific treefrog (Hyla regilla), whose populations are not known to be in decline. The Western toad (Bufo boreas) and the Cascades frog (Rana cascadae), whose populations have declined markedly, showed significantly lower photolyase levels. In field experiments, the hatching success of embryos exposed to UV radiation was significantly greater in H. regilla than in R. cascadae and B. boreas. Moreover, in R. cascadae and B. boreas, hatching success was greater in regimes shielded from UV radiation compared with regimes that allowed UV radiation. These observations are thus consistent with the UV-sensitivity hypothesis.

Mechanisms and ecological correlates of kin recognition.

Kin recognition, which can be inferred from differential interactions amongst close relatives as compared to unrelated conspecifics, has been documented in a wide range of vertebrates and invertebrates. Kin may be discriminated by phenotypic traits (signatures) that are genetically mediated, acquired from the environment, or arise from an interaction between these two sources. The developmental mechanisms underlying recognition of relatives (or their signatures) include direct contact and familiarization, indirect familiarity (whereby previously unencountered individuals are recognized by their resemblance to familiar kin) and hypothetical recognition alleles. The biological significance and mediating mechanisms of kin recognition vary across species according to their ecology and natural history. To illustrate this point, the results of laboratory and field studies of larvae of several species of toads and frogs are reviewed.

An investigation of the alarm response in Bufo boreas and Rana cascadae tadpoles.

Tadpoles of the western toad (Bufo boreas) and of the Cascades frog (Rana cascadae) show an alarm reaction to an extract containing chemical cues from damaged conspecifics. The mean time spent by individual B. boreas tadpoles in the half of the test tank to which the extract solution was added was significantly lower than expected by chance. Activity was also significantly greater in Bufo extract tests than in control tests. Tadpoles did not avoid an extract of another tadpole species (Hyla regilla). Rana cascadae tadpoles did not avoid areas containing Rana extract but did significantly increase their level of activity in response to the extract. These results suggest that the R. cascadae tadpole alarm reaction exists but differs from the B. boreas reaction.

Kin recognition in Rana cascadae tadpoles: effects of rearing with nonsiblings and varying the strength of the stimulus cues.

Experiments with two clutches of Rana cascadae tadpoles were undertaken to determine if (1) rearing tadpoles exclusively with nonkin affects sibling recognition behavior and (2) if tadpoles are capable of distinguishing between groups of varying sibling/nonsibling composition. Individual tadpoles reared from embryonic stages with 12 nonsiblings behaved as tadpoles reared with siblings by exhibiting a preference for siblings over nonsiblings in choice tests. This suggests that experience with siblings is not a necessary prerequisite for the development of sibling recognition in this species. Tadpoles reared with siblings preferred to associate with a group composed of 50% siblings and 50% nonsiblings over a group of 100% nonsiblings, but exhibited no preference for a group composed of 25% siblings and 75% nonsiblings versus one composed of nonsiblings only. Thus tadpoles are capable of discriminating between mixed groups of siblings and nonsiblings, but probably require some minimum proportion of siblings to do so.

Kin recognition cues in Rana cascadae tadpoles.

The importance of chemical, visual, and auditory cues in kin recognition behavior in Rana cascadae tadpoles was tested in laboratory choice experiments. Tadpoles were given a choice of associating with siblings or nonsiblings by using visual and chemical stimuli together (standard tests), visual stimuli alone, or chemical stimuli alone. We also monitored sound production of tadpoles. We conclude that tadpoles can distinguish between siblings and nonsiblings by using waterborne chemical cues alone that are probably sensed by olfaction or taste. Visual stimuli alone are not sufficient for such discrimination and sound production as a means of communication in tadpoles is probably unimportant.