EcoHealth 3, 5–21, 2006 DOI: 10.1007/s10393-005-0006-4
2006 EcoHealth Journal Consortium
Original Contributions
Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus (Batrachochytrium dendrobatidis) Cynthia Carey,1 Judsen E. Bruzgul,1 Lauren J. Livo,1 Margie L. Walling,2 Kristin A. Kuehl,1 Brenner F. Dixon,1 Allan P. Pessier,3 Ross A. Alford,4 and Kevin B. Rogers5 1
Department of Integrative Physiology, University of Colorado, Boulder, CO 80309-0354 Department of Environmental and Radiological Health Sciences/Epidemiology, Colorado State University, Ft. Collins, CO 80521 3 Department of Pathology, Zoological Society of San Diego, PO Box 120551, San Diego, CA 92112-0551 4 School of Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia 5 Colorado Division of Wildlife, PO Box 775777, Steamboat Springs, CO 80477 2
Abstract: One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 107–108 zoosporangia before death results. Variation in air temperature between 12C and 23C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified. Key words: Batrachochytrium dendrobatidis, amphibian pathogen, Bufo boreas, chytrid fungus, chytridiomycosis
INTRODUCTION Populations of many species of amphibians have experienced serious declines over the last several decades (Alford and Richards, 1999; Stuart et al., 2004). Although factors such as habitat destruction and introduction of invasive species have contributed to these declines, infectious disease has been identified as another significant cause (Berger et al., 1998; Carey, 2000; Carey et al., 1999, 2003a, 2003b; Published online: January 18, 2006 Correspondence to: Cynthia Carey, e-mail:
[email protected]
Daszak et al., 1999, 2003). A recently discovered chytrid fungus (Batrachochytrium dendrobatidis), initially isolated from captive amphibians suffering from a mycotic skin disease (Longcore et al., 1999; Nichols et al., 1998; Pessier et al., 1999), has now been linked to mass mortalities of wild amphibian populations in many areas, including Europe, South America, Central America, Australia, New Zealand, and North America (Berger et al., 1998; Bishop, 2000; Bosch et al., 2001; Bradley et al., 2002; Green et al., 2002; Green and Kagarise Sherman, 2001; Lips et al., 2003; Muths et al., 2003; Ron et al., 2003; Ron and Merino, 2000). For a recent list of geographic localities and amphibian species on
6 Cynthia Carey et al.
which this chytrid fungus has been found, see Carey et al., (2003a) or Speare and Berger (2000). This pathogen, thought to have recently emerged (Daszak et al., 2003), demonstrates little genetic diversity among isolates collected in various locations around the world (Morehouse et al., 2003). The closest chytrid relative from which this pathogenic form evolved is currently not known (James et al., 2000). Although the geographic origin of B. dendrobatidis has not been proven, evidence from museum specimens suggests it may have originated in Africa (Weldon et al., 2004). A wide variety of bacterial, viral, and fungal agents are normally found on the skin, in the digestive tract, and in other tissues of amphibians (Granoff, 1969; Taylor, 2001; Taylor et al., 2001). However, this chytrid fungus and a group of ranaviruses are the only pathogens for which there is a demonstrated correlation between the degree of infection observed in laboratory animals and similar degrees of infection from animals captured in nature, including those from populations experiencing mass mortality events and population declines (Carey et al., 2003a, 2003b; Green et al., 2002; Jancovich et al., 1997; Nichols et al., 2001). Although most members of the phylum Chytridiomycota typically meet their nutritional requirements by breaking down organic matter in aquatic systems, some species are parasitic upon selected invertebrates, such as insects (Longcore et al., 1999). B. dendrobatidis is the first chytrid fungus known to be parasitic upon a vertebrate host. Zoospores of this fungus preferentially attack keratinocytes in the skin of metamorphosed amphibians (Longcore et al., 1999; Pessier et al., 1999). Although amphibian larvae appear to lack keratin in their epidermis, this pathogen has been identified in the keratinized mouthparts of tadpoles and toes of premetamorphic tadpoles of a few species (Berger et al., 1998; Carey et al., 2003a, 2003b; Fellers et al., 2001; Rachowicz and Vredenburg, 2004; Marantelli et al., 2004). In the few years since its discovery, some progress has been made in understanding the interactions of this fungal pathogen with amphibians (see reviews by Berger et al., 2004; Carey et al., 2003a, 2003b; Daszak et al., 2003; Davidson et al., 2003; Longcore et al., 1999; McDonald et al., 2005; Nichols et al., 2001; Pessier et al., 1999; Retallick et al., 2004). Nichols et al., (2001) experimentally exposed captive Dendrobates tinctorius and D. auratus to the type isolate of B. dendrobatidis by dripping a solution containing zoospores onto the back and legs of these amphibians once a day for 30 days or 4–5 days per week for 4 weeks or by
immersing them in water containing chytrid zoospores in their cages. All exposed animals died with skin infections, whereas control animals did not develop infections. Some amphibian species, such as bullfrogs (Rana catesbeiana) and tiger salamanders (Ambystoma tigrinum), apparently carry the pathogen on their epidermis without developing lethal infections; therefore, they can serve as reservoirs for the transmission of chytridiomycosis to susceptible species (Daszak et al., 2003; Davidson et al., 2003; Hanselmann et al., 2004; Weldon et al., 2004). Further, B. dendrobatidis can persist in an endemic state in healthy frogs once an epidemic wave has passed through amphibian populations (McDonald et al., 2005; Retallick et al., 2004). To date, however, many questions remain about the interaction of this pathogen with amphibians, including what constitutes the minimal infective dose of zoospores necessary to cause a lethal infection, how various environmental factors affect the ability of zoospores to cause an infection, and how various amphibian species differ in susceptibility to infection by this pathogen. Much more information about these interactions is needed to develop effective methods for preventing further amphibian mortalities and population declines. We here report on a series of experiments designed to examine the interaction of B. dendrobatidis with the boreal toad (Bufo boreas), a species that is known to be susceptible to infection by this pathogen. Boreal toads, widely distributed throughout many parts of the western United States, suffered severe population declines and extinctions in the southeastern part of their range (Carey, 1993; Corn et al., 1989) in the late 1970s to early 1980s. As a result, this species is classified as ‘‘endangered’’ in Colorado (Goettl, 1997) and New Mexico, a ‘‘species of special concern’’ in Wyoming (Keinath and Bennett, 2000), and a ‘‘sensitive species’’ in Utah (Utah Division of Wildlife Resources, 1998); it is classified as ‘‘warranted but precluded’’ for federal listing as a threatened or endangered species by the U.S. Fish and Wildlife Service. Since 1996, B. dendrobatidis has been associated with mortalities in some of the few remaining populations of toads in Colorado (Muths et al., 2003). Its presence in a few museum specimens collected in Colorado during the 1970s and the similarities between the patterns of historical declines and current mass mortalities suggest, but do not conclusively prove, that the historical die-offs of this species in Colorado in the 1970s were due to this pathogen (Carey, 1993; Carey et al., 1999). The boreal toad is an ideal model system for examining the interactions between Batrachochytrium and amphibians because
Exposure of Boreal Toads to B. dendrobatidis
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chytrid-free boreal toadlets can be obtained from a Colorado Division of Wildlife hatchery and because an isolate of this fungal pathogen from a boreal toad in a Colorado population is available for experimentation. These experiments were designed to investigate (1) whether lethal infections due to chytridiomycosis can be induced experimentally in boreal toadlets, (2) how variations in temperature and body mass affect survival of boreal toadlets exposed to B. dendrobatidis zoospores, (3) whether uninfected boreal toadlets can become infected by exposure to water in which B. dendrobatidis-infected toadlets have been housed, and (4) how the duration of exposure to, and the dosage of B. dendrobatidis zoospores affect the postexposure survival of boreal toadlets. Answers to these questions are important for identifying factors that might contribute to the ability of B. dendrobatidis to infect and kill boreal toads in nature. Although the interactions between zoospores and amphibians may differ in nature and in the laboratory, experimental measurements of the number of zoospores necessary to initiate a fatal infection and the amount of exposure time necessary for infection to occur, as well as experimental determination of whether or not amphibians can become infected by contact with water containing zoospores in the laboratory, are valuable steps in understanding these phenomena in nature. In addition, it is important to understand how environmental factors, such as temperature, affect the dynamics of infection of amphibians by B. dendrobatidis.
after the experiments described here were conducted, we were unable to verify that all the toadlets were free of chytrid infection at the start of each experiment. However, the toadlets were raised by the hatchery in a manner that minimized exposure to B. dendrobatidis and no outbreaks of this pathogen have been reported for hatchery toads. Subsequent PCR analysis of samples from the hatchery indicates that the hatchery currently is free from contamination with this pathogen. Therefore, we feel confident that the toadlets had not been exposed to B. dendrobatidis prior to their use in these experiments. After transport from NASRF to the University of Colorado in Boulder, young-of-the-year toadlets were maintained at 23C, the temperature shown to be optimal for B. dendrobatidis growth (Piotrowski et al., 2004), on a 12-hour/12-hour light/dark cycle in troughs containing tap water. Toadlets were fed Drosophila or pin-head crickets (Fluker Farms, Port Allen, LA) three times a week. The crickets were fed Fluker’s Calcium Fortified Cricket Quencher in order to provide vitamins and minerals to the toadlets. During experiments, toadlets were housed in walk-in environmental chambers in which temperature, light, and feeding conditions were controlled as described above, unless specified otherwise in individual experiments. In each experiment, however, toadlets were held in 20% Holtfreter’s solution (pH 6.5, the optimal pH for B. dendrobatidis growth [Piotrowski et al., 2004]), made with double-distilled water to minimize exposure of chytrid zoospores to contaminants in tap water.
MATERIALS
B. dendrobatidis Cultures and Exposures
AND
METHODS
General Husbandry Because boreal toads are designated as endangered or threatened throughout the southeastern part of their geographic distribution, our experiments were necessarily restricted to toadlets that were provided from the Colorado Division of Wildlife’s John W. Mumma Native Aquatic Species Restoration Facility (NASRF). The toadlets used in these experiments were raised at NASRF from eggs or tadpoles collected at five different localities in the mountains of Colorado in late May through June. Because of the relative synchronicity of egg laying at various boreal toad breeding locations, the age of toadlets used in this study probably differed by less than 6 weeks, regardless of the locality of origin. Because the development of a polymerase chain reaction (PCR) assay for B. dendrobatidis occurred
Isolate JEL#275 of B. dendrobatidis was used to expose toadlets to this pathogen. This strain was originally isolated from an infected boreal toad from Clear Creek County, Colorado, by Joyce Longcore. Cultures were grown in Hbroth (10 g tryptone and 3.2 g glucose/liter distilled water) at 23C for 4–7 days prior to the beginning of an experiment. On the day that toadlets were to be exposed to B. dendrobatidis, zoospores were filtered through a sterile 20 lm nylon mesh filter (Spectra/Mesh; Spectrum Laboratories, Rancho Dominguez, CA) to remove sporangia and then an aliquot of the filtrate was counted with a hemacytometer to determine the concentration of zoospores per milliliter in the filtrate. A solution containing the desired concentration of zoospores was made by diluting the filtered H-broth culture with 20% Holtfreter’s solution (pH 6.5) and sufficient penicillin/streptomycin (Sigma, St. Louis,
8 Cynthia Carey et al.
MO) to comprise 1% of the final volume. Toads in the exposure groups were placed in aliquots of this solution. Because the toadlets were exposed to the chytrid zoospores in a nonsterile environment, penicillin/streptomycin was necessary to minimize bacterial growth on the nutrients in the H-broth. Toads in control groups were exposed to identical proportions of B. dendrobatidis-free H-broth and penicillin/streptomycin in 20% Holtfreter’s solution.
Detection of B. dendrobatidis DNA in Toad Skin These experiments were conducted before a PCR test for B. dendrobatidis DNA was developed. In anticipation of the development of this test, samples were collected during or at the end of these experiments by scraping the ventral skin with a sharpened wooden dowel, which was then placed in 1 ml of 0.25 M EDTA (pH 8) saturated with NaCl. These samples were subsequently analyzed by Pisces Molecular (Boulder, CO) for DNA specific to B. dendrobatidis using PCR primers developed by Annis et al., (2004). Toad carcasses were stored in 10% formalin. Sections of skin from the ventral pelvic region (pelvic or drink patch) were processed for histological examination and embedded in paraffin. Sections (5 lm) were stained with hematoxylin and eosin.
Experiment 1: Can Infections with Batrachochytrium Be Experimentally Induced in Boreal Toadlets, and How Do Temperature and Body Mass Affect the Survival of These Toadlets after Exposure? Our first experiment was designed to test whether boreal toadlets could be infected experimentally with the B. dendrobatidis isolate JEL#275. Each of 160 toadlets was weighed, placed in an individual 236 ml plastic Ziploc container with a perforated lid, and randomly assigned to one of four groups: control or exposed, each maintained at either 23C or 12C. Mass ranged from 1.6–38.0 g (average ± standard error [SE] mass = 12.41 ± 0.56 g). Analysis of variance (ANOVA) revealed no significant differences in mean initial mass for toadlets among groups (F = 0.170, P = 0.6806) or between the two temperature treatments (F = 2.324, P = 0.1294). The group · temperature interaction was also nonsignificant (F = 44.701, degree of freedom [DF] = 1, P = 0.3430). For toadlets in exposure groups, we used what we believed to be a large dose (106 zoospores/toadlet daily) and
a relatively long exposure time (72 hours) to maximize the chance of producing skin infections and mortality under laboratory conditions. Toads in the exposure groups were exposed individually to this dose of zoospores in 20 ml of Holtfreter’s solution constituted as described above. This volume was adequate to cover the floor of the Ziploc container and to immerse the ventral side of the toadlet throughout the exposure period. Control toadlets were individually exposed to an identical volume of the sham exposure solution. Fresh solutions containing zoospores were made from B. dendrobatidis cultures each day. After 24 and 48 hours, the initial exposure solutions were discarded from each container and replaced with the same volume of identically constituted solutions. After 72 hours, all toadlets were transferred from the small containers and placed individually in 15 · 30 · 11 cm plastic containers with 200 ml of B. dendrobatidis-free 20% Holtfreter’s solution (pH 6.5). Holtfreter’s solutions in the cages were replaced three times per week when the toadlets were fed. The containers were held flat so that the ventral surfaces of the toadlets in a normal sitting posture were in continuous contact with the solution. In order to examine the time course between exposure and the appearance of detectable infection, five toadlets from each group were sacrificed by cervical dislocation, followed by spinal and cerebral pithing, on a random sampling schedule on postexposure days 1, 3, 8, 14, and 21. Skin scrapes were collected for PCR analysis, and carcasses were preserved for histological examination. Thirty-two of the 80 exposed toadlets died on or prior to their assigned sampling date. On the assumption that these toadlets died from B. dendrobatidis infections (assessed after death with histology and, later, PCR), experiment 1 afforded additional opportunities to analyze the effects of two variables, temperature variation and toadlet body mass, that could plausibly affect the number of days to death. The relation of the number of days to death as a function of body mass was evaluated with least-squares regression. The average time of survival of toadlets that died prior to their scheduled sampling date in two temperature treatments, 23C and 12C, was evaluated using Student’s t-test. Tissue samples from all toadlets that died prior to their scheduled sampling date were analyzed by PCR. As the experiment progressed, we noted several behavioral and physiological differences between control and exposed toadlets at each temperature. Anecdotal observations of behavioral differences were not statistically
Exposure of Boreal Toads to B. dendrobatidis
analyzed. However, on the last sampling date, the number of respirations per minute of all surviving exposed toadlets (n12C = 6, n23C = 5) and randomly selected control toadlets (n12C = 6, n23C = 4) were counted; the mean number of respirations per minute of control and exposed animals was compared with two-way ANOVA, with treatment (control or exposed) and temperature (12C or 23C) as the independent variables. When the experiment was terminated 3 weeks after the initial exposure, some control and all of the remaining exposed animals were sacrificed. Holtfreter’s solution and the containers in which exposed and control animals had been sitting for 24 hours were immediately used in experiment 2.
Experiment 2: Can Uninfected Boreal Toadlets Become Infected by Exposure to Liquid (i.e., Holtfreter’s Solution) in Which Infected Toadlets Have Been Housed? This experiment was designed to determine whether chytridiomycosis could be transmitted to uninfected animals through exposure to Holtfreter’s solutions used in the previous experiment to house infected toadlets. Twentyone control animals remained at the end of experiment 1: 11 had been maintained at 12C and 10 at 23C. These toadlets were assigned randomly to containers from experiment 1 which had housed either control or exposed toadlets in 200 ml Holtfreter’s solution that had been in the container the preceding 24 hours. Toadlets for experiment 2 remained in the experiment 1 Holtfreter’s solution for 48 hours, after which the liquid was replaced with clean, B. dendrobatidis-free, 20% Holtfreter’s solution (pH 6.5). This solution was then changed three times per week during feedings. Survival of toadlets was monitored daily for 34 days. When a toadlet died, the date of death was recorded and skin scrapes were collected for PCR analysis. A logrank test on censored survival data (StatXact; Cytel Statistical Software, Cambridge, MA) was used to assess whether the patterns of survival of control and exposed groups differed significantly. Body masses of toadlets used this experiment ranged between 6 and 30 g. Average body masses of control and exposed toadlets, compared using two-way ANOVA, did not differ significantly (P = 0.6782, DF = 1) and there was no significant interaction term (P = 0.6636, DF = 1). However, the average mass of the combined control and exposed toadlets at 23C was significantly heavier than the control and exposed body masses at 12C.
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Experiments 3 and 4: How Do the Time of Exposure and the Dosage of B. dendrobatidis Zoospores Affect the Survival of Boreal Toadlets? As noted below (see Results), PCR analysis and histology of tissues from exposed toadlets in experiment 1 indicated that exposure to 106 zoospores per day for 3 days was sufficient to cause chytridiomycosis and death. Experiment 3 examined the effects of a variety of dosages and durations of exposure on the number of days survived. The experiment was designed as a 2 · 5 factorial, with one factor being exposure duration (1 or 3 days) and the other being dosage of zoospores/20 ml (0 [control], an estimated 1 zoospore, 100, 104, or 106 zoospores], resulting in a total of 10 experimental groups with 15 toadlets each. Toadlets were weighed and randomly assigned to one of the 10 groups. Mean initial toadlet mass did not differ significantly among groups (F = 0.576, DF = 9, P = 0.8154). Individual toadlets from control and varying dosage groups designated for 1-day exposure were placed in 236 ml Ziploc containers containing 20% Holtfreter’s solution (pH 6.5) with either B. dendrobatidis-free broth (controls) or broth containing various concentrations of zoospores for 24 hours, after which the solutions were replaced daily with 20 ml Holtfreter’s solution (pH 6.5). After 3 days, toadlets were placed in individual 15 · 30 · 11 cm plastic boxes for the duration of the experiment. Even though these toadlets were exposed to B. dendrobatidis zoospores for only 1 day, this procedure on days 2 and 3 ensured that handling of the toadlets in the 1-day and 3-day exposures was identical. Toadlets that were exposed for 3 days and their controls were kept in the small Ziploc containers for 3 days and given fresh B. dendrobatidis-containing or B. dendrobatidis-free doses of Holtfeter’s solution daily. At the end of the 3-day exposure, these groups were also placed individually in large plastic boxes at 23C for the 42-day duration of the experiment. Toads were fed and water was changed three times per week; mortality was monitored daily. Skin scrapes for PCR analysis were collected at death or, for toadlets that survived the entire 42-day period, at the end of the experiment. In experiment 4, we examined the effect of 1-day exposure to a different set of zoospore concentrations (0 [control], 20, 40, 60, 100, and 103 zoospores/20 ml) on survivability with groups of 10 Bufo boreas toadlets each. This experiment was also conducted at 23C and monitored for 42 days under the same housing and care conditions as in experiment 3. No significant differences in
10 Cynthia Carey et al.
initial toadlet mass existed among groups (F5,54 = 1.725, P = 0.1445). At the end of the experiment, skin scrapes were collected for PCR analysis. Survival curves of control and exposure groups in experiments 3 and 4 were evaluated by the StatXact logrank test for censured survival data. This test indicated whether the rates of death and percentage of animals in each group that died differed significantly among dosages and (in experiment 3) the duration of exposure. Additionally, the known fate modeling procedure in program MARK (White and Burnham, 1999) was used to isolate the most parsimonious models from suites of candidate models containing all possible first-order combinations of dose, exposure duration (in experiment 3), and body mass to determine their relative importance in determining the length of survival. Program MARK uses second-order Akaiki information criteria (AICc; Burnham and Anderson, 2002) to identify the model that best describes the data without sample overparameterization. Using this approach for model selection is superior to traditional hypothesis testing for this data set because it allows simultaneous comparison of multiple candidate models, balances precision and bias when selecting the appropriate model (Burnham and Anderson, 2002), and is not restricted to parametric data. In addition, we examined whether the results of these experiments were consistent with a simple model of the disease process, based on the assumption that populations of B. dendrobatidis on the host grow exponentially until a threshold density is reached that produces host mortality. Exponential population growth can be represented as a linear increase of log(number of parasites) with time. A linear function has two parameters, the slope and the intercept. The intercept for these population growth lines is the number of zoospores that settled on the first day of exposure, day 1 of this experiment. For treatments in which toadlets were exposed to an estimated single zoospore, this must equal log10(1) for successful infections. For other treatments, this number should be equal to log10(np), where n is the number of zoospores to which the toadlets in each treatment were exposed and p is the proportion of zoospores that colonize a toadlet in 1 day of exposure. For experiment 3, we assumed that p was constant across all treatments, estimated p using two methods, and used the average of these estimates for our model. One estimate of p was calculated as the proportion of toadlets that were exposed to an estimated single zoospore for 1 day that became infected. The second was calculated for the treatment in
which individuals were exposed to 100 zoospores as the probability of colonization per zoospore that made the zero term of the binomial distribution equal to the observed proportion of failures to infect. We also assumed that all population growth lines had the same slopes. If all the growth lines had the same slopes, differing only in intercepts, and mortality occurs when these growth lines cross a threshold number, then the common slope of the growth lines can be estimated by dividing 2 (the difference in elevation in log units between the lines for initial exposures to 102, 104, and 106 zoospores) by the differences in time to mortality averaged between all possible pairs of individuals in the treatments exposed for 1 day to 102 or 104 and to 104 or 106 zoospores. We compared the average time to mortality predicted by these four lines with the observed time to mortality for toadlets exposed to zoospores for 1 day. We cross-checked these results with those for individuals exposed to zoospores for 3 days. We assumed that the slopes and intercepts of the B. dendrobatidis population growth lines remained the same as those for individuals exposed for 1 day. We calculated the total estimated B. dendrobatidis population on individuals in each treatment as the sum of the three population growth lines for the populations started on each day of exposure and determined estimated times to mortality by inspecting the values of these sums. We also examined how well our growth models fit the data from experiment 4. We assumed that the slopes of population growth lines remained the same as those estimated for the third experiment since the conditions under which toadlets were maintained following infection were identical. We estimated the intercepts of the growth lines for each exposure level in the same manner but used a value of p derived from the observed rates of infection in this experiment, by calculating the p values necessary to make the zero term of the binomial distribution equal to the observed proportion of failures to infect for the 20, 40, and 60 zoospore treatments and averaging these values.
RESULTS Experiment 1: Can Infections with B. dendrobatidis Be Experimentally Induced in Boreal Toadlets, and How Do Temperature and Body Mass Affect the Survival of These Toadlets after Exposure? Both histological examination of ventral skin and PCR analysis for B. dendrobatidis DNA in ventral skin confirmed that exposure of boreal toadlets to 106 zoospores for 3 days
Exposure of Boreal Toads to B. dendrobatidis
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Figure 1. Histopathology of Bufo boreas experimentally infected with B. dendrobatidis. A: Skin from an uninfected control, magnified ·40. B: Skin from a toad exposed to B. dendrobatidis 12 days previously. The epidermis is thickened with disorganized keratinocytes (hyperplasia), and there is a cluster of chytrid thalli within the superficial keratinized layers (stratum corneum, center), magnified ·40. C: Detail of chytrid thalli within the stratum corneum. Numerous developmental stages are present including a flaskshaped zoosporangium containing numerous discrete zoospores.
caused chytridiomycosis. Histological lesions observed in exposed toadlets were moderate epidermal hyperplasia characterized by increased epidermal thickness and disorganization of keratinocytes and mild to moderate orthokeratotic hyperkeratosis (Fig. 1). Within keratinocytes in the stratum corneum, there were moderate numbers of characteristic chytrid thalli, including zoosporangia with developed zoospores and septate (colonial) thalli characteristic of B. dendrobatidis. Chytrid thalli were evident in histological sections as early as 9 days following infection. Tissues of only 3 of the 80 exposed animals tested negative for B. dendrobatidis DNA; these were sampled either on day 1 or day 3 following exposure. Additionally, 32 exposed animals died on or prior to their scheduled sampling date. Because both histological analysis and PCR indicated the presence of B. dendrobatidis in the skin of these animals, we concluded that 3-day exposure to 106 zoospores was sufficient to cause death in boreal toadlets. One control toadlet died on the first day of the 21day experiment and was found to be free of B. dendrobatidis infection by PCR analysis. Tissues of 6 of the 20 control toadlets submitted for PCR analysis were weakly positive for B. dendrobatidis DNA, probably due to contamination during the sampling procedure at the end of the study. Although instruments were cleaned with ethanol between samples, this cleaning procedure may not have been sufficient to remove all B. dendrobatidis DNA, resulting in contamination. No other control animals, including those toadlets nominally positive for B. dend-
robatidis DNA, died prematurely before their assigned sampling date. The mean number of days survived by toadlets that died before their scheduled sampling date was 14.0 ± 0.8 days (n = 14) and 13.8 ± 0.8 days (n = 18) at 12C and 23C, respectively. These values did not differ significantly (t = 0.188, P = 0.8525, DF = 30), indicating that temperature variation over this range had no significant effect on the survival time of these boreal toadlets. Body mass of all toadlets in experiment 1 ranged between 1–38 g, but the masses of those that died on or before their scheduled sampling date ranged only between 4–19 g. Our regression analysis indicated that toadlet body mass within this range had a significant effect on the number of days survived. Because temperature had no significant effect on survival time, the days of survival of toadlets held at 12C and 23C were pooled and analyzed; a highly significant correlation existed between the number of days survived following exposure to B. dendrobatidis zoospores and toadlet body mass (r = 0.795, n = 30, P < 0.0001; Fig. 2). The least-squares regression equation best describing this relationship is as follows: Days survived = 8.79 + 0.545 · Mass (g) (F = 49.798, P < 0.0001). An SAS t-test (Satterthwaite method) showed a highly significant relationship between mass and survival time, with shorter survival times associated with smaller masses (t = 3.62, P = 0.0018). Control and exposed toadlets exhibited several behavioral differences as the experiment progressed. Specifically, exposed toadlets held their bodies out of water as
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Figure 2. Number of days survived by boreal toadlets (Bufo boreas) as a function of body mass following exposure to 106 zoospores of B. dendrobatidis for 3 days. Each point represents data for one toad.
Table 1. Mean Breaths per Minute of Control (Nonexposed) Boreal Toadlets (Bufo boreas) or Toads Exposed to Chytrid Fungal Zoospores (Batrachochytrium dendrobatidis) at Two Constant Temperatures Treatment and temperature
Mean ± SE breaths/min
n
Control (12C) Exposed (12C) Control (23C) Exposed (23C)
147 125 195 128
6 6 4 5
± ± ± ±
5.4 9.1 9.0 24.5
much as possible by climbing on the walls of their container or by adopting a four-legged posture that raised their ventral surface above the water on the bottom of the cage. Sometimes exposed toadlets elevated their toes out of the water. In comparison, control toadlets usually sat in the water and were rarely observed with ventral body surfaces out of the water. ANOVA indicated that control toadlets had a significantly higher respiration rate than exposed toadlets by the end of the 3-week experiment (F = 10.6, DF = 1, P = 0.0047; Table 1). Temperature had no significant effect on respiration rates of exposed and control toadlets held at 12C and 23C (F = 3.592, DF = 1, P = 0.0752), nor did the interaction between treatment and temperature (F = 2.770, DF = 1, P = 0.1144).
Figure 3. Changes in the percentage survival of groups of boreal toadlets (Bufo boreas) as a function of time (days) during a 34-day experiment following placement in water in which uninfected toads (control group) or toads infected with B. dendrobatidis (exposed group) had been held. n = 10 for controls (5 at 23C, 5 at 12C), n = 11 for exposed (5 at 23C, 6 at 12C).
Experiment 2: Can Uninfected Boreal Toadlets Become Infected by Exposure to Water in Which Infected Toadlets Have Been Housed? Exposure to water in which toadlets infected with B. dendrobatidis had been living for 24 hours caused significant chytridiomycosis and death of boreal toadlets (Fig. 3). A log-rank test for censored survival data indicated a significant difference in the pattern of survival of control and
Exposure of Boreal Toads to B. dendrobatidis
13
Figure 4. Experiment 3: Changes in the percentage survival of groups of boreal toadlets (Bufo boreas) exposed to different dosages of B. dendrobatidis zoospores for 1 or 3 days in a 42-day experiment. n = 15 for each group.
exposed toadlets (two-sided exact inference P = 0.0001). Mortalities of exposed toadlets began on day 16. Exposed toadlets held at 12C and 23C survived an average of 23.8 ± 2.42 (n = 6) and 25.45 ± 2.56 (n = 4) days, respectively, during the 34-day experiment. Only one of the exposed toadlets (at 23C) survived the full 34 days of the experiment. All of the exposed toadlets tested positive for B. dendrobatidis DNA by PCR analysis. In contrast, 9 of the 10 toadlets (5 at 12C and 5 at 23C) transferred to water previously used by control (nonexposed) toadlets survived the entire 34 days of the experiment. One control toadlet (at 23C) died on day 34. None of the control toadlets, including the one that died on day 34, tested positive for B. dendrobatidis DNA. During the course of this experiment, toadlets placed in water previously occupied by B. dendrobatidis-exposed toadlets exhibited avoidance of water and decreased rates of respiration as noted in exposed toadlets from experiment 1.
Experiments 3 and 4: How Do the Time of Exposure and the Dosage of B. dendrobatidis Zoospores Affect the Survival of Boreal Toadlets? Both the number of zoospores to which a toadlet was exposed (dosage) and the number of days of exposure strongly affected the duration of survival of exposed boreal toadlets (log-rank test two-sided exact inference P = 0.0001). The average number of days survived by toadlets exposed to 106 zoospores (15.5 ± 1.9 and 16.4 ± 3.9 days for the 1- and 3-day exposure groups, respectively) did not differ significantly (Fig. 4). At lower dosages (an estimated 1, 100, or 10,000 zoospores), both dosage and duration of
exposure to B. dendrobatidis zoospores significantly affected the mean length of survival, percent mortality, and percentage of toadlets actually infected by the exposure treatment (Table 2). The duration of exposure had no effect on the percentage of toadlets surviving at high dosages: all toadlets in groups exposed to 10,000 and 106 zoospores died by the end of the test (Table 2). At lower dosages, only 13% of toadlets exposed for 3 days to an estimated single zoospore survived, whereas 93% of those exposed to that dosage for only 1 day survived the 42-day experiment (Table 2). Finally, both the time of exposure and the dosage affected the percentage of toadlets in each group infected by B. dendrobatidis. For example, only 38% of the toadlets became infected when exposed to an estimated single zoospore for 24 hours, yet 100% were infected by a 3-day exposure (Table 2). Duration of exposure, dose, and toadlet mass were factors available for analysis for their effects on survival time in experiment 3. These factors were evaluated by ranking a suite of candidate models in program MARK using the logit link function (Table 3). The model that included exposure duration and dose (model A) had nearly three times the support (AICc weight = 0.572) than the one that also included mass (model B, AICc weight = 0.225). Of the top four models (models A-D), those that included exposure duration as a variable (A and B) were almost four times more strongly supported than those without (C and D). Dose was selected in all four of the top models, demonstrating its importance in predicting survival of toadlets. The most parsimonious model was as follows:
14 Cynthia Carey et al.
Table 2. Proportion (%) of Toadlets Surviving 42 Days following Exposure to Varying Dosages of Chytrid Fungi Zoospores and Proportion That Tested Positive for B. dendrobatidis by PCR: Control Groups Were Not Exposed to the Fungus Experiment 3 dosages (n = 15)
Control
1
100
10,000
1,000,000
100% 0%
13% 100%
0% n/a
0% n/a
0% n/a
100% 0%
93% 38%
27% n/a
0% n/a
0% n/a
Experiment 4 dosages (n = 10)
Control
20
40
60
100
1000
1-Day exposure Surviving 42 days Positive
100% 10%
50% 100%
60% 60%
40% 90%
30% 90%
0% 100%
3-Day exposure Surviving 42 days Positive 1-Day exposure Surviving 42 days Positive
n/a, not applicable.
Table 3. Models Considered by Program MARK to Predict Survival (S) in Toadlets from Experiment 3 and Their Ranking by AICc: Parameters Considered Include Exposure Duration (Dur), Dose, and Mass
Model
Parameters
AICc
Delta AICc
AICc Weight
Model Likelihood
Par
Deviance
A B C D E F G
S(DurDose) S(DurDoseMass) S(Dose) S(DoseMass) S(Dur) S(DurMass) S(.)
835.965 837.832 838.727 840.476 913.565 915.209 917.353
0.00 1.87 2.76 4.51 77.60 79.24 81.39
0.572 0.225 0.144 0.060 0.000 0.000 0.000
1.0000 0.3933 0.2514 0.1048 0.0000 0.0000 0.0000
3 4 2 3 2 3 1
829.960 829.823 834.724 834.470 909.562 909.203 915.352
Par, number of parameters; S(.), daily survival, was the only parameter used in the model.
S ¼
expð4:987 þ 0:459DUR 0:684DOSEÞ 1 expð4:987 þ 0:459DUR 0:684DOSEÞ
where S is daily survival, DUR is the exposure duration, and DOSE is the dose used. Toadlets were exposed to 100 zoospores for 1 day in both experiments 3 and 4. The percentage of toadlets surviving the 42-day experiments was similar (27% in experiment 3 and 30% in experiment 4). A t-test showed no significant difference in mean survival time for toadlets at this common dosage level (mean survival(experiment 3) = 34.5 ± 1.6 days, mean survival(experiment 4) = 33.0 ± 2.3 days; t = 0.562, P = 0.5796, DF = 23). These results give us confidence that the conditions under which the experiments were run, although offset in time, were sufficiently
similar that the results can be compared between the two experiments. Except for the 100-zoospore exposure replicate, toadlets were exposed to a different set of dosages in experiment 4. All toadlets exposed to 1,000 zoospores died within 42 days. At dosages lower than 1,000 zoospores, the percentage of toadlets surviving the 42-day experiment and the percentage infected with B. dendrobatidis varied among groups (Table 2, Fig. 5); some toadlets exposed to lower dosages lived the full 42 days (Fig. 5, Table 2). One control animal also tested weakly positive for B. dendrobatidis DNA (Table 2). A similar suite of candidate models were ranked by program Mark for experiment 4 (Table 4). Because experiment 4 had only a single exposure duration and the doses were within an order of magnitude, the contribution
Exposure of Boreal Toads to B. dendrobatidis
15
Figure 5. Experiment 4: changes in the percentage survival of groups of boreal toadlets (Bufo boreas) exposed to different dosages of B. dendrobatidis zoospores for 1 day in a 42-day experiment. n = 10 for each group.
Table 4. Models Considered by Program MARK to Predict Survival (S) in Toadlets from Experiment 4 and Their Ranking by AICc: Parameters Considered Include Dose and Mass
Model
Parameters
AICc
Delta AICc
AICc Weight
Model Likelihood
Par
Deviance
H I J K
S(DoseMass) S(Mass) S(Dose) S(.)
304.472 304.797 307.117 308.280
0.00 0.33 2.65 3.81
0.441 0.375 0.118 0.066
1.0000 0.8497 0.2664 0.1489
3 2 2 1
298.455 300.789 303.108 306.278
Par, number of parameters; S(.), daily survival, was the only parameter used in the model.
of mass was more than three times that for dose in terms of toadlet survival (model I AICc weight = 0.375 vs. model J AICc weight = 0.118). Even though a small range of masses was used in this experiment, a size-related effect on survival was clearly demonstrated, with increasing mass resulting in increased survival time. This mass effect can be summarized as follows: S ¼
expð2:568 þ 0:155MASSÞ 1 þ expð2:568 þ 0:155MASSÞ
For model I, S is daily survival and MASS is given in grams. We used the data from experiments 3 and 4 to address the question of whether death occurs when the number of B. dendrobatidis sporangia infecting a toadlet exceeds a threshold. Our models of the growth of B. dendrobatidis populations on individuals fit the data closely (Table 5) and predict that the threshold for death is about 107–108 zoosporangia per toadlet. Using the models fitted for 1-day exposures in experiment 3 produced predicted times to
mortality that were close to the actual times experienced by individuals exposed to zoospores over 3 days. The intercepts estimated for experiment 4 using the proportion of animals successfully infected are low, suggesting that rates of successful establishment of zoospores on hosts were lower in this experiment than in experiment 3. However, the models also fit the data for this experiment reasonably well. The relationship between observed and predicted median dates of mortality for experiments 3 and 4 is shown in Figure 6. The fact that the regression accounts for most of the variation in the data and has a slope almost identical to 1 suggests that our models are a good reflection of the actual disease process in boreal toads.
DISCUSSION We believe that this study has a number of significant findings: (1) chytridiomycosis can be experimentally in-
16 Cynthia Carey et al.
Table 5. Data and Parameters of Models of Exponential Amphibian Chytrid Population Growth on Hosts Fitted to Data from Experiments 3 and 4a Number of zoospores Days exposed Experiment 3 1 1 1 1 3 3 3 3 Experiment 4 1 1 1
Time to death (days)
Population growth line
n
Log10(n)
Median
Mean difference
Predicted
Slope
Intercept
Log10(estimated fatal threshold)
1 100 10,000 1 · 106 1 100 10,000 1 · 106
0.000 2.000 4.000 6.000 0.000 2.000 4.000 6.000
31 27 22 14 26 23 17 15
2.909 6.860 6.697
27 25 18 12 27 24 18 11
0.295 0.295 0.295 0.295 0.295 0.295 0.295 0.295
0.000 0.665 2.665 4.665 0.000 0.665 2.665 4.665
8.851 8.336 8.861 8.501 7.622 7.403 7.633 9.042
20 40 60
1.301 1.602 1.778
28 25.5 24.5
27 27 27
0.295 0.295 0.295
0.072 0.044 0.117
8.333 7.715 7.493
2.445 4.919 3.510
5.050 0.583 -3.976
a Fatal threshold is the number of B. dendrobatidis estimated to be present on hosts at the median day of mortality for each treatment. Mean difference represents the average of all pairwise differences in time to mortality between individuals in a treatment and individuals in the treatment in the same experiment with the next higher number of zoospores per 20 ml. Fatal threshold B. dendrobatidis numbers represent the number calculated to be in the average population on hosts in the given treatment at the median day of mortality; for hosts exposed over 3 days, these are the sum of the three growth curves for those initial populations.
Figure 6. Modeled survival by mass of boreal toadlets at low dosages ranging from 20 to 100 zoospores of B. dendrobatidis. Comparison of observed median days survival postinfection of infected animals in each treatment in experiments 3 and 4, with median days survival predicted using models assuming that amphibian chytrid populations grow at equal rates on all hosts and produce mortality when a threshold number per host is reached. Dashed line indicates line of equality, solid line is regression line, y = 0.994x – 1.02.
duced in boreal toadlets; (2) the duration of exposure and the dosage of B. dendrobatidis play important roles in determining the length of survival; (3) our model predicts that the level of infection must reach a threshold to cause death; (4) larger toadlets live longer given a particular dosage than smaller toadlets, at least within the
size range of toadlets used in this study; (5) housing exposed animals at air temperatures of 12C and 23C has no significant effect on the length of survival following exposure; and (6) lethal chytridiomycosis can be transmitted through the water in which infected toadlets have been sitting. Following a discussion of each of these
Exposure of Boreal Toads to B. dendrobatidis
findings, the findings of this study will be correlated with observations of the population biology of boreal toad populations experiencing mass mortalities associated with B. dendrobatidis.
Chytridiomycosis Can Be Experimentally Induced in Boreal Toadlets This study showed that lethal chytridiomycosis can be experimentally induced in the laboratory in boreal toadlets by exposure to B. dendrobatidis zoospores and, in most instances, death followed exposure within 5–7 weeks. In our first experiment, exposure to 106 zoospores for 72 hours caused infections in 96% of boreal toadlets in that group. These results coupled with those of Nichols et al., (2001) indicate that chytridiomycosis can be induced readily in susceptible amphibian species. Behavioral and physiological changes in the toadlets were noted as the experiment progressed. First, infected toadlets tried to avoid contact with water in the bottom of their cages. The avoidance of water, regardless of the motivational cause underlying the behavior, should prolong survival following infection because the rate of reinfection from zoospores released from the skin surface would be low whenever the ventral surface was dry. Second, the rate of respirations decreased as the severity of the infections increased. These results may reflect a gradual inhibition of metabolism by pathological changes in the skin caused by B. dendrobatidis. These observations do not support the hypothesis that B. dendrobatidis kills amphibians by blocking oxygen uptake through the skin. If this hypothesis were true, lung ventilation should increase if cutaneous respiration is curtailed by B. dendrobatidis infection. However, our data show the opposite trend.
Duration of Exposure and Dosage of B. dendrobatidis Play Important Roles in the Length of Survival The results of experiments 3 and 4 indicate that the initial dosage of zoospores to which toadlets were exposed and the length of exposure have significant effects on the number of toadlets infected and the number of days survived (Figs. 4 and 5). Low dosages and a 1-day exposure fostered longer average survival times than high dosages and a 3-day exposure duration. However, average mortality of groups exposed to 1 or 3 days of the highest dosage level of 106 zoospores did not differ significantly. The significance of these data will be discussed in the next section.
17
Although toadlets were exposed for 24 hours to their individual dose of zoospores in a small (20 ml) amount of solution, not all individuals became infected at low exposures. Our estimates of initial sizes of B. dendrobatidis populations on toadlets (Table 5) suggest that approximately 4% of the zoospores present in the solution used for exposures became established on the host during 24 hours in experiment 3, while only 1% became established during 24 hours of exposure in experiment 4. Therefore, it appears that a relatively high proportion (96–99%) of the zoospores present in the immediate vicinity of toadlets fail to colonize them successfully during a 24-hour period.
Reinfection by Zoospores Released by an Individual Is Necessary for the Level of Infection to Reach a Threshold Necessary to Cause Death In experiment 1, both PCR and histological analysis of toadlets killed at intervals throughout the experiment showed increases in the amount of B. dendrobatidis present and indicated that death occurred with moderate to heavy infections. In this study, even a nominal dose of 1 zoospore in the water may be sufficient to result eventually in a lethal infection. Our data show an inverse correlation between dosage and length of survival: the higher the number of zoospores in the initial exposure, the shorter the time to death. The finding that the number of days survived by boreal toadlets following exposure to B. dendrobatidis zoospores is directly related to dosage supports the contention that heavy infections are needed to cause mortality. Exposure to a few zoospores causes infection, but time is needed for multiple reinfections to reach the threshold required to kill the animal. Exposure to a large number of zoospores causes death more rapidly than a small number because a large number of zoospores can relatively quickly produce the critical degree of infection necessary to cause death. Similarly, the length of exposure is also important for reaching the critical level of infection to cause death. With the exception of toadlets infected with 106 zoospores, the average days of survival was lower in toadlets exposed for 3 days than for 1 day for a given dosage. Our growth model (Fig. 6) indicates that the approximate threshold for infection to cause death ranges between 107 and 108 zoosporangia (thalli). When a zoospore invades amphibian skin, it invades a keratinocyte and transforms into a sporangium, which produces zoospores. Mature zoospores exit from the sporangium through a discharge tube into the
18 Cynthia Carey et al.
water adjacent to the epidermis. The zoospores then can reinfect the same host or find other hosts. The mechanism by which B. dendrobatidis kills amphibians is as yet unknown, but two hypotheses have been advanced (Berger et al., 1998; Carey et al., 2003a) that are consistent with the idea that a certain level of infection must be reached to cause death. First, the fungus might produce a toxin that diffuses from the epidermis into the body and causes lethal tissue damage. A heavy concentration of sporangia would produce more toxin than a light one. Second, the presence of this chytrid in the epidermis causes hyperplasia and hyperkeratosis, or excess production of skin layers. Thickening of the skin layers in the ventral skin of the pelvic area is hypothesized to cause lethal disruptions in water and ion balance. A heavy infection would result in a greater degree of ionic and/or water imbalance than a light one.
Larger Toadlets Will Live Longer Given a Particular Dosage than Smaller Toadlets, at Least within the Size Range of Toadlets Used in This Study Body size was a significant factor in determining the length of survival of an infected toadlet in experiments 1, 3, and 4. The size range of toadlets used in these experiments does not represent the total variation in body mass observed in field populations because these toadlets were young of the year. Some boreal toads, particularly females, can grow to over 80 g (Carey et al., 2005), but the endangered status of this toad precludes use of large boreal toads in laboratory experiments. Therefore, the significant association between the duration of survival of boreal toadlets and body mass may not necessarily hold for larger toads. It is unknown why smaller animals died more rapidly than larger ones. We have demonstrated that exposing toadlets of similar sizes to different doses of zoospores leads to differences in time to death consistent with the existence of a threshold number of thalli necessary to cause death. It seems likely that the size of this threshold is directly related to the surface area of the animal. The threshold of small animals, therefore, is both smaller and reached more quickly than the threshold for larger animals. The effect may relate to the fact that small toadlets have a relatively small surface area of ventral skin. Alternatively, the skin of smaller toadlets might be easier to penetrate than that of larger ones. Also, there might be size-, and therefore age-, related differences in maturation of skin immune defenses, such as the ability to synthesize and secrete antimicrobial peptides.
Housing Exposed Animals at Air Temperatures of 12C and 23C Has No Significant Effect on Length of Survival Following Exposure Numerous anecdotal accounts suggest that temperature can be an important cofactor contributing to the success of B. dendrobatidis in causing mortality of amphibians. Specifically, a number of amphibian deaths linked to B. dendrobatidis coincide with the onset of seasonally cold temperatures (Berger et al., 2004; Carey, 2000; Carey et al., 1999; McDonald et al., 2005; Retallick et al., 2004). Further, studies of a tropical frog indicate that B. dendrobatidis infection may be cured by exposure to a temperature of 37C (Woodhams et al., 2003). These previous studies document that very high and very low temperatures affect the interaction between B. dendrobatidis and its hosts. This study, conducted at moderate temperatures, found that housing exposed animals at 12C or 23C had no significant effect on survival in both experiments 1 and 2. Most physiological and cellular rate processes increase two- to threefold for every 10C rise in temperature (the Q10 rule) (Rome et al., 1992). Several important factors could be temperature-dependent, including the growth rate of sporangia, the rate of zoospore release, and the swimming speed, penetration rate, and keratinocyte invasion rate by zoospores. However, this study shows that the overall length of time survived by B. dendrobatidis-infected boreal toadlets does not obey the Q10 rule. Additional research on the effect of temperature on the interactions between B. dendrobatidis and its hosts is needed.
Lethal Chytridiomycosis Can Be Transmitted through the Water in Which Infected Boreal Toadlets Have Been Sitting Experiment 2 confirms the findings of Marantelli et al., (2004) that direct body contact is not necessary for chytridiomycosis to be transmitted. Within 21 days after infection with 106 zoospores per day for 3 days, toadlets shed sufficient zoospores into Holtfreter’s solution to kill previously uninfected toadlets. A recent study indicates that water can remain infective for up to 7 weeks after the introduction of B. dendrobatidis (Johnson and Speare, 2003). Therefore, this pathogen, shed by an infected individual in a breeding pond in June, could be transmitted to an uninfected individual coming into contact with the water at this location for much of the summer activity period, even if no other infected amphibians were present.
Exposure of Boreal Toads to B. dendrobatidis
Correlations of the Findings of This Study with Observations on Populations of Boreal Toads in Colorado Experiencing Mass Mortalities Associated with B. dendrobatidis These studies were conducted in environmental conditions that differed considerably from those in the habitat of boreal toads, and as a result, they cannot be used to predict the survival times of boreal toads infected with B. dendrobatidis in nature. The conditions in these experiments were optimal for B. dendrobatidis: 23C and pH 6.5 are within the optimal ranges for growth of this fungus in laboratory culture (Piotrowski et al., 2004). Furthermore, in this study, toads were held in continuous contact with water. Since toads can reinfect themselves with zoospores presumably only when they are in water or on a moist substrate, constant contact with water undoubtedly promoted reinfection and shortened the survival time relative to the probable survival time if the toads had been periodically allowed to dry their skin. In contrast, boreal toad body temperatures, the pH of the water with which they come into contact, and the amount of daily contact with water vary considerably in their native habitat in ways that should promote longer survival times of infected toads in the field than in these experimental conditions. Body temperatures of boreal toads fluctuate between near freezing and almost 30C daily during their 3- to 4-month summer activity period (Carey, 1978). Toads spend the other 7–8 months of each year in hibernation under snow and ice, where their body temperatures remain near freezing. The pH of the water in which these toads come into contact varies widely from location to location and throughout the year (Jones et al., 2001). Finally, nonbreeding individuals of this species spend at least part of each day during their summer active period away from contact with water. Despite these differences, the results of this study can contribute to our understanding of some of the observations that we have made on boreal toad populations experiencing mass mortalities associated B. dendrobatidis. During these events, population sizes decrease either to extinction or to very low levels (Carey, 1993; Muths et al., 2003). We have noticed that larger toads (adults) tended to persist in the population longer than smaller ones (postmetamorphic juveniles) and that adult females tended to survive more years than adult males following the initial documentation of the presence of B. dendrobatidis and subsequent mortality of most toads in the population.
19
The range of body sizes used in this study was small compared to the full range (up to more than 80 g) of body masses found in the field (Carey et al., 2005). Because approximately 4–6 years are required to grow to adult body size, typical boreal toad populations have a broad spectrum of body sizes. Although further study is necessary to verify that body size has an effect on survival in large boreal toads, our data are consistent with observations that juveniles and smaller males died out before larger females in infected populations (Carey, personal observation). We presume that most boreal toads become infected with B. dendrobatidis in aquatic environments, such as breeding ponds, hibernacula, or moist substrate on which adults sit and bask in the sun during the summer. This pathogen, shed by an infected individual breeding in June, could be transmitted to an uninfected individual coming into contact with the water in this location for much of the summer active period, even if no other infected amphibians were present. The number of zoospores released in the field from an infected boreal toad is not known, but we anticipate that a lightly infected individual would shed fewer zoospores than a heavily infected one. Therefore, the dosage to which an uninfected animal would be exposed in the field would vary with the number of infected individuals present, the severity of their infections, the proximity of the healthy individual to the infected one, the life span of zoospores, and probably other factors. Adult females exhibit several behaviors that could minimize their risk of infection by B. dendrobatidis zoospores and that support our observations that adult females in infected populations may live longer generally than adult males during an outbreak of B. dendrobatidis. Adult males are likely to spend several weeks during breeding each spring in continuous contact with water and to have frequent skin-to-skin contact with other adult males. The risk of infection in breeding ponds is minimized for females because they spend less than 1 day at a breeding site during egg laying and because they do not breed every year (Carey et al., 2005). Females may also minimize the risk of infection by hibernating more frequently as solitary individuals than males, some of whom have been observed to hibernate communally (Carey, personal observation). Most boreal toad populations in Colorado became extinct in the late 1970s through early 1980s (Carey, 1993; Corn et al., 1989). One hypothesis to explain why the few relict populations survived these mass extinction events was that they might have possessed some sort of immune resistance against whatever pathogen might have caused the mass
20 Cynthia Carey et al.
mortalities, thought at the time to be Aeromonas hydrophila (Carey, 1993). Although the exact causes of the 1970s mortalities will never be proven conclusively, the presence of chytridiomycosis in museum specimens collected in Colorado during the mid-1970s and the similarities between the pattern of mass mortalities in the 1970s and 1990s (the latter known to be linked to B. dendrobatidis; Muths et al., 2003) suggest that this pathogen was the infectious agent (Carey et al., 1999). Toadlets in this study originated from five populations located in different mountain ranges in the Colorado Rockies. Because individuals from each of these populations proved to be susceptible to this pathogen under the conditions of these experiments, we conclude that the persistence of these relict populations was not likely related to immunity to this pathogen.
Acknowledgments This study was supported by a National Science Foundation grant (Integrated Research Challenges in Environmental Biology, DEB-0213851). We thank the Colorado Division of Wildlife, especially Mark Jones, Chuck Loeffler, and Craig Fetkavich, for facilitating use of animals from the John W. Mumma Native Aquatic Species Restoration Facility for this research. Joyce E. Longcore generously provided B. dendrobatidis cultures and advice throughout the course of these experiments. John Wood of Pisces Molecular, Inc. made several recommendations concerning PCR sampling, for which we are grateful.
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