Abstract
Emerging fungal pathogens pose a greater threat to biodiversity than any other parasitic group1, causing declines of many taxa, including bats, corals, bees, snakes and amphibians1–4. Currently, there is little evidence that wild animals can acquire resistance to these pathogens5. Batrachochytrium dendrobatidis is a pathogenic fungus implicated in the recent global decline of amphibians6. Here we demonstrate that three species of amphibians can acquire behavioural or immunological resistance to B. dendrobatidis. Frogs learned to avoid the fungus after just one B. dendrobatidis exposure and temperature-induced clearance. In subsequent experiments in which B. dendrobatidis avoidance was prevented, the number of previous exposures was a negative predictor of B. dendrobatidis burden on frogs and B. dendrobatidis-induced mortality, and was a positive predictor of lymphocyte abundance and proliferation. These results suggest that amphibians can acquire immunity to B. dendrobatidis that overcomes pathogen-induced immunosuppression7–9 and increases their survival. Importantly, exposure to dead fungus induced a similar magnitude of acquired resistance as exposure to live fungus. Exposure of frogs to B. dendrobatidis antigens might offer a practical way to protect pathogen-naive amphibians and facilitate the reintroduction of amphibians to locations in the wild where B. dendrobatidis persists. Moreover, given the conserved nature of vertebrate immune responses to fungi5 and the fact that many animals are capable of learning to avoid natural enemies10, these results offer hope that other wild animal taxa threatened by invasive fungi might be rescued by management approaches based on herd immunity.
In recent decades, emerging fungal pathogens have had devastating effects on agricultureand caused population declines of several plant and animal species1. Many plants can acquire immunological resistance to fungal pathogens, which has proven useful for managing fungal pathogens of crops11, but acquired resistance to fungi in wild animals has not been well studied5.If natural variation in acquired resistance to fungi exists in wild animal populations, it might partly explain why fungal pathogens cause epidemics and extirpations of some animal populations (those that have not acquired resistance) but persist in an endemic state in others (those that have acquired resistance)12,13. If wildlife managers could induce resistance in enough individuals of a population, they might be able to drive the basic reproductive ratio (R0; the average number of infections one infected individual generates in a population of susceptible hosts over the course of its infectious period) of a pathogenic fungus below one, such that ‘herd immunity’ would protect even pathogen-naive members of the population, a concept that is the basis for vaccination campaigns. Consequently, acquired resistance offers a potential tool to rescue animal populations threatened by fungi14.
Here, we investigate whether amphibians can acquire behavioural and immunological resistance to the fungal pathogen Batrachochytrium dendrobatidis (Bd). We define acquired resistance broadly as the process of reducing infection loads upon subsequent exposure to a pathogen or antigen caused by changes in behaviour, innate immunity or adaptive immunity within the lifetime of an individual. We focused on Bd because it is implicated in the global decline of amphibians, the most threatened vertebrate taxon6. Bd is known to hinder amphibian lymphocyte responses7, but acquired resistance might still be detectable if its strength exceeds any immunosuppression by Bd. Hence, it is critical to quantify the net effect of any acquired host resistance and Bd-induced immunosuppression on Bd abundance (average number of Bd zoospores on Bd-exposed hosts) across multiple host species to evaluate the feasibility of acquired resistance as a conservation tool14. Although beneficial for understanding mechanisms of immunity, gene expression studies or studies that only quantify immunity to Bd(and not also Bd loads)do not capture the extended phenotype and thus cannot quantify the net effect of acquired immunity and immunosuppression on the host–parasite interaction8,9,15,16. Surprisingly, there are no published studies that experimentally subjected amphibians to regimes of repeated Bd infection followed by clearance and then tested for an association between the number of previous infections and Bd abundance, immune parameters and behavioural avoidance (ref. 14, but see also 15–22). We hypothesized that, despite Bd-induced immunosuppression7–9, effects of repeated exposures to Bd followed by pathogen clearance could include reduced Bd abundance on frog skin, increases in the abundance or efficacy of skin peptides, augmentation of the abundance of responding lymphocyte populations (mediators of Bd resistance16–18), and induction of learned avoidance of Bd.
To create variation in number of exposures to Bd, groups of frogs were exposed to Bd and cleared of their infection using heat zero to four times (depending on their treatment assignment and experiment) in a manner that prevented a simple decay of immune responses from accounting for our results (Extended Data Table 1; Supplementary Methods). Frogs were swabbed for Bd before each experiment, after each Bd exposure, and after each clearance period. Quantitative PCR (qPCR) of these swabs revealed that frogs were free of Bd before each experiment, that all unexposed control frogs were Bd-free after each Bd growth period (that is, no cross contamination), that exposed frogs generally became infected (average prevalence across exposure periods was 85%), and that heat-clearances were 100% effective at eliminating established Bd infections (Extended Data Table 2). Additionally, our methods ensured that we did not confound acquired resistance, a form of phenotypic plasticity, with selection (via mortality of low-resistance individuals; see Supplementary Results, Extended Data Table 3, Extended Data Fig. 1) or Bd inoculates (via different inoculates for each treatment), confounding factors that have hampered previous studies14.
Many hosts are capable of avoiding pathogens, but few studies have tested whether avoidance is learned or innate23,24. To determine if amphibians could learn to avoid Bd, we conducted two separate experiments to examine how number of exposures to Bd affected the amount of time that oak toads (Bufo quercicus) spent on a Bd-negative or Bd-inoculated side of a test chamber. Bd-naive frogs showed no significant avoidance or attraction to Bd (χ21 = 0.289, P = 0.591), but frogs previously infected with Bd once or twice (all the exposures resulted in infections) chose the Bd-free substrate approximately 65% and 70% of the time, respectively (experiment 1: χ21 = 6.683, P = 0.009, experiment 2: χ21 = 9.693, P = 0.002; Fig. 1), resulting in a significant interaction between naivety and Bd avoidance (number of previous infections × deviation from 50% null interaction: χ21 = 9.107, P = 0.011; Fig. 1). These results were consistent across the two experiments (that is, repeatable; Fig. 1). Importantly, this learned behavioural resistance should be unaffected by the immunosuppressive effects of Bd7–9.
Figure 1. Effects of 0, 1 or 2 previous exposures to live Batrachochytrium dendrobatidis (Bd) on the proportion of time that toads (Bufo quercicus) spent on the Bd-positive side of a test chamber in two experiments (Experiment 1: circles, Experiment 2: squares).
In both experiments (n = 30 toads), Bd-naive frogs showed no significant avoidance or attraction to Bd (χ21 = 0.29, P = 0.59), but frogs previously infected with Bd once or twice chose the Bd-free substrate more frequently than expected by chance (χ21 = 6.7, P = 0.009 and χ21 = 9.7, P = 0.002, respectively). Asterisks represent significant avoidance of Bd.
To test for acquired immunological resistance, we assessed whether the number of exposures to live Bd (0, 1, 2, 3 or 4 exposures) was a significant predictor of Bd abundance on Cuban treefrogs (Osteopilus septentrionalis) after the third and fourth Bd exposure and clearance regimens (Extended Data Table 1). Number of exposures to Bd was a significant negative predictor of Bd abundance on the frogs (χ21 = 8.40, P = 0.003; Fig. 2a and Extended Data Fig. 2a; see Extended Data Table 3 for prevalence data), with a 75% drop in Bd loads from the first to third Bd exposure (Fig. 2a, open circles). To evaluate whether the observed resistance to Bd could be attributable to changes in innate or cellular immunity, we quantified the abundance and efficacy of skin peptides and splenic leukocytes (enriched for lymphocytes) after only the fourth exposure and clearance (0–4 exposures) and then tested whether number of Bd exposures was a significant predictor of these responses. The observed acquired resistance to Bd did not seem to be strongly attributable to changes in skin peptides because the number of Bd exposures was not significantly correlated with skin peptide abundance (χ21 = 0.7, P = 0.38; Extended Data Fig. 3a) or efficacy at inhibiting Bd growth in vitro (Peptide treatment × number of Bd exposures: χ21 = 0.329, P = 0.566; Extended Data Fig. 3b, Supporting Information). However, number of Bd exposures was a significant positive predictor of lymphocyte abundance in the spleen (χ21 = 5.9, P = 0.015, Fig. 3a) and lymphocyte proliferation in response to Bd (χ21 = 9.5, P = 0.002) and phytohaemagglutinin (χ21 = 78.4, P < 0.0001; number of exposures × method of stimulation: χ21 = 1.3, P = 0.24; Fig. 3b). Phytohaemagglutinin is a mitogen that triggers T-lymphocyte cell division and thus functioned as a positive control for lymphocyte proliferation25. Moreover, the slope of the relationship between Bd abundance and lymphocyte numbers in the spleen became more positive with each Bd exposure, indicating that lymphocytes were stimulated to proliferate with subsequent exposures (number of exposures × lymphocyte abundance: F1,54 = 4.4, P = 0.04, Fig. 3c).
Figure 2. Effects of 1–4 exposures to Batrachochytrium dendrobatidis (Bd) on Bd abundance per gram of frog and frog (Osteopilus septentrionalis) survival.
a, b, Effects of exposures on mean Bd abundance (zoospore genome equivalents (GE)/g of frog) after exposure period 3 (± s.e.m., open circles and left axis; χ21 = 8.40, P = 0.003) and exposure period 4 (closed circles, right axis; bootstrapped means ± bootstrapped 95% confidence interval, see Supplementary Methods for details) in the first immunological resistance experiment (a) and exposure period 4 in the second immunological resistance experiment ( ± s.e.m.; live Bd exposures (circles and solid line): χ21 = 4.9, P = 0.02; dead Bd exposures (squares and dotted line): χ21 = 11.3, P < 0.001) (b). c, Effects of live Bd exposures on mean frog survival ( ± 95% confidence interval; odds ratio: 1.66, χ21 = 4.45, P = 0.035; experiment treated as a temporal block). The best-fit lines are based on predicted values from the implemented zero-inflated negative binomial (Bd abundance) and binomial statistical models (frog survival). Naivety was based on the state of the frog before Bd exposure during the focal exposure period (third or fourth exposure period depending on what is being displayed). Thus, frogs exposed to Bd for the first time during the focal exposure period were classified as naive because they had not previously been exposed to Bd.
Figure 3. Effects of 0–4 exposures to live Batrachochytrium dendrobatidis (Bd) on immune parameters of frogs (Osteopilus septentrionalis).
a, Mean (± s.e.m.) lymphocyte abundance in the spleen (that is, splenocytes) per gram body weight (gbw). Splenocyte densities did not differ within the naive (χ21 = 0.35, P = 0.56) or experienced groups (χ21 = 0.14, P = 0.93) but differed significantly between these two groups (χ21 = 11.35, P < 0.001; designated by the horizontal lines). Frogs exposed to Bd for the first time during the fourth exposure period were classified as naive because they had not previously been exposed to Bd. b, Lymphocyte proliferation when cultured as splenocytes alone (squares and solid line), or with heat-killed Bd zoospores (circles and dashed line) or phytohaemagglutinin (PHA; diamonds and dotted line). Values provided are predicted values of proliferated lymphocytes (measured as counts per minute by a scintillation counter) based on a negative binomial model (n = 19, 17, 17, 10 and 7, respectively for 0–4 previous live Bd exposures for both the splenocytes alone (that is, control) and splenocytes plus live Bd and n = 15, 13, 11, 2 and 4, respectively, for splenocytes plus PHA). See text for statistics. c, Slope (± s.e.) of the relationship between Bd abundance and splenocyte abundance (F1,54 = 4.4, P = 0.04), showing greater lymphocyte proliferation in response to Bd load with each previous Bd exposure.
Two previous studies attempted to immunize frogs systemically through injections and did not find evidence of acquired protection against Bd21,26, suggesting that the approach used in our study, exposure by way of the skin, might be critical for induction of acquired resistance. Consistent with this proposition are studies suggesting that the adaptive immune system of amphibians responds to Bd. For example, the heterozygosity of genes associated with adaptive immunity (major histocompatibility complex loci) was greater in frog populations more resistant to Bd18, which would have been exposed cutaneously. Additionally, frogs irradiated to reduce their lymphocyte numbers had increased susceptibility to Bd infections, and frogs immunized against Bd via injection with heat-killed Bd cells had elevated levels of Bd-specific IgM and IgY serum antibodies17.
The only previous study to repeatedly infect and clear amphibians of Bd seemed to conflict with our results20. In this study20, the authors concluded that there was no evidence of acquired resistance in the critically endangered booroolong frog (Litoria booroolongensis) because frogs previously cleared of Bd with the fungicide itraconazole did not have significantly lower prevalence or mortality upon re-infection than frogs that were not previously exposed to Bd or the fungicide (20 of 32 versus 14 of 28 infected, respectively; χ21 = 0.9, P = 0.33). However, the fungicide alone increased Bd prevalence in Bd-naive frogs (no previous fungicide: 14 of 28 infected, previous fungicide: 10 of 11 infected, χ21 = 5.5, P = 0.01), consistent with other studies suggesting that itraconazole is immunosuppressive27. Hence, a better test for acquired resistance would have been to compare Bd prevalence of frogs previously infected and cleared of Bd by the fungicide to the prevalence of Bd-naive frogs also previously exposed to the fungicide. A re-analysis of these data show that previous infections significantly reduced prevalence from 91% to 63% (χ21 = 3.1, P = 0.03) when making this more direct comparison. In summary, despite the immunosuppressive effects of Bd7–9, the net effect of previous exposures to and clearances of Bd, across three studies and three species (B. quercicus, O. septentrionalis, L. booroolongensis), was to reduce Bd prevalence or abundance on frogs. Differences in the strength of this net effect (immunosuppression + acquired resistance) or variation in pathogenicity across Bd strains might explain host variation in susceptibility to Bd21,26.
Next, we sought to test whether amphibians could acquire resistance to dead Bd because induction of acquired resistance through exposure to dead Bd might offer a practical management tool to protect amphibian populations14. We repeated our first immunological resistance experiment described above but added a dead Bd exposure treatment and tracked frog survival for 6 weeks after the last Bd inoculation to enhance our survival estimates. Number of exposures to live Bd in this second immunological resistance experiment was again a significant negative predictor of Bd abundance on O. septentrionalis (χ21 = 4.9, P = 0.02; Fig. 2b, Extended Data Fig. 2b), replicating our previous findings. Additionally, when blocking by experiment (P < 0.001), number of exposures to live Bd was a significant positive predictor of the probability of surviving the experiment (logistic regression: χ21 = 4.4, P = 0.03; Fig. 2c) and a positive predictor of time of death (Cox survival analysis: χ21 = 4.6, P = 0.03, parameter ± s.e. = 0.224 ± 0.105, hazard ratio = 0.799; Supplementary Fig. 3). Indeed, frogs with previous exposure to Bd were 5.57 (odds ratio) times more likely to survive until the end of the experiment than frogs that were naive to Bd.
Similar to our findings for exposure to live Bd, number of previous exposures to dead Bd was a significant negative predictor of Bd abundance on frogs (χ21 = 11.3, P < 0.001) and the magnitude of acquired resistance to dead and live Bd did not significantly differ (χ21 = 0.99, P = 0.32; Fig. 2b, Extended Data Fig. 2b). By the end of six weeks of Bd growth, frogs previously exposed to dead Bd three or four times lived longer than those exposed to dead Bd two times (df = 2, Wald = 7.22, P = 0.027), but overall, number of exposures to dead Bd was not a significant predictor of time of death (χ21 = 0.02, P = 0.900; Extended Data Fig. 4). Our power for detecting an effect of dead Bd on survival, however, was lower than it was for live Bd because we only conducted one rather than two experiments with dead Bd.
As a result of efforts by the IUCN Amphibian Ark network and other conservation initiatives, hundreds of threatened amphibian species have been removed from their Bd-positive habitats and are being bred in captivity14,28. However, these amphibians often failto re-establish when released at their sites of collection, presumably because of the persistence of Bd at these sites on tolerant hosts14,28,29. Inducing acquired resistance in these captive-bred amphibians might allow for their successful re-establishment14,28. Although additional research is necessary to quantify the efficacy of releasing dead Bd into water bodies to protect amphibians and the non-target effects of these releases, mathematical models suggest that, despite biotic and abiotic reservoirs for Bd, this strategy offers a promising management tool to reduce R0 of Bd below one, which could be used to proactively prevent Bd epidemics and to rescue host populations already threatened by this pathogen12,13. However, the efficacy of this management option will depend on several factors, such as whether Bd-naive larval amphibians can also acquire immunity, the role of biotic and abiotic reservoirs in maintaining Bd, and the extent and magnitude of variation in acquired resistance among amphibian species. Perhaps most importantly, given the conserved nature of the immune responses of vertebrates to fungi5 and that many animals are capable of learning to avoid natural enemies10, the results presented here offer hope that other wild animal taxa threatened by invasive fungi, such as bats, bees, and snakes1–4, might be capable of acquiring resistance and might also be rescued by management approaches based on herd immunity.
METHODS
Bd culture and inoculation
For all experiments we prepared Bd inoculum (strain SRS 812) according to the methods used in McMahon et al.28 (see Supplementary Methods for additional details).
First and second immunological resistance experiments
We collected O. septentrionalis eggs from Bd-free wading pools at the University of South Florida Botanical Gardens (Tampa, FL) and reared them through metamorphosis in a Bd-free laboratory. All frogs were approximately five months post-metamorphosis before the first and second immunological resistance experiments were initiated and all frogs were assigned randomly to treatments so that time since metamorphosis should not be confounded with number of Bd exposures. For the first immunological resistance experiment, we exposed frogs to and cleared frogs of Bd 0–4 times (n = 10–20/treatment; see Extended Data Table 3 for sample sizes). After each exposure and clearance period, each frog was weighed and swabbed 10 times from hip to toe (left leg after Bd growth period, right leg after clearance period) to determine the abundance of Bd via qPCR. Frogs were fed crickets ad libitum, mortality was monitored daily, and moist paper towels were changed weekly. These methods were repeated in the second immunological resistance experiment, except that frogs were exposed to live or flash frozen (culture in flask placedin liquid nitrogenfor 15 min) Bd 0–4 times (n = 20 per treatment; Extended Data Tables 1, 2). For each exposure period, we plated 1 ml of the previously frozen Bd inoculum on three 1% tryptone agar plates and incubated each at 23 °C for at least 8 days (see Supplementary Methods). No Bd grew confirming that the Bd was successfully killed. Frogs were exposed to dead Bd every 2 days for 11 days to match the live Bd exposure in this same experiment (see Supplementary Methods and Supplementary Results for a preliminary experiment demonstrating that dead Bd DNA was not detectable after 2 days). In the first immunological resistance experiment, frogs were cleared after the last 11-day Bd exposure period to ensure that we had adequate frog survival and sample sizes for immune assays. In the second immunological resistance experiment, we quantified frog survival for six weeks after the last live Bd inoculation so we could better assess the effects of acquired resistance on survival. In the fourth exposure period after exposure to dead Bd, frogs were challenged with live Bd to test whether previous exposure to dead Bd induced acquired resistance. In the first immunological resistance experiment, we quantified Bd abundance on frogs after each exposure period but only present the data for exposure periods three and four. In the second immunological resistance experiment, we did not conduct the qPCR to quantify Bd loads in the third exposure period because the dead Bd treatment had not yet received the live Bd challenge. Thus, we only conducted the qPCR for the fourth exposure period in this experiment.
Antimicrobial skin peptide and splenic lymphocyte collection and assays
In the first immunological resistance experiment, we used the methods of Rollins-Smith et al.25,30 to quantify skin peptide abundance and efficacy at inhibiting Bd growth and to quantify lymphocyte abundance in the spleen and lymphocyte proliferation in response to freshly killed Bd zoospores (60 °C 10 min) and phytohaemagglutinin (see Supplementary Methods for additional details).
Behavioural resistance experiment
We field collected adult oak toads (B. quercicus; n = 30, Extended Data Table 3) from Hillsborough County, FL and reared and monitored them individually in the laboratory as described in the first immunological resistance experiment. Toads were placed in a test chamber (9.53 × 7.0 cm) containing two 3.03 × 9.5 cm paper towels on each side of the chamber separated by a 1.0 cm gap. We randomly dosed one side with 2.0 ml of a Bd+ inoculum and the other side with 2.0 ml of Bd− inoculum. We then placed a toad in the centre of the chamber, allowed it to acclimate for 30 min, and then conducted double blind scan samples every 5 min for 40 min, recording the side of the chamber containing each toad. After the behavioural trials, the toads were transferred to new containers and experienced the Bd exposure-clearance regime described in the first immunological resistance experiment. Behavioural observations were repeated after each clearance period so that by the end of behavioural resistance experiment 1 and 2, we quantified toad avoidance of Bd after 0 or 1 and 0, 1, or 2 infections, respectively (a dysfunctional environmental chamber caused a mass mortality event preventing the second exposure for experiment 1).
Quantitative PCR
We followed the procedure described by Hyatt et al.30 to quantify Bd abundance using qPCR (with a StepOne Real-Time PCR System; Applied Biosystems, Foster City, CA). See Supplementary Methods for additional details.
Statistical analyses
All animals were randomly assigned to treatments, statistics were analysed with R statistical software, significance was attributed when P < 0.05, frog mass was used a covariate when significant (unless the response was mass standardized), and all analyses were conducted within rather than across exposure periods so that treatment comparisons were made among frogs exposed to the same Bd inoculate. For the first and second immunological resistance experiments, the effect of number of live Bd exposures on survival was analysed using Cox proportional hazards regression (package: survival, function: coxph), blocking by experiment. A generalized linear model (package: stats, function: glm), was used to determine whether number of Bd exposures affected frog growth, lymphocyte densities, and skin peptide abundance. A mixed effects model (package: nlme, function: lme) was used to test for the main and interactive effects of skin peptides (presence/absence) and number of Bd exposures on Bd growth (defined as(ln(OD7) – ln(OD0))/7 where OD7 and OD0 refer to optical density measurements at 490 nm on days 7 and 0, respectively) in culture, treating frog identity as a random effect (see Supplementary Information). A mixed effects negative binomial model (package: glmmADMB, function: glmmadmb) was used to determine if there was a difference in lymphocyte proliferation in response to Bd and PHA for each frog (individuals frogs treated as a random effect) compared to their lymphocyte proliferation in the absence of either stimulus. A general linear model was used to test whether number of Bd exposures was a predictor of the slope of the relationship between Bd abundance and lymphocyte densities. We used a zero-inflated negative binomial error model (package: pscl, function: zeroinf) to test for the effect of number of exposures on Bd abundance within the third exposure period for the first immunological resistance experiment and within the fourth exposure period for the second immunological resistance experiment. Bootstrapping analyses were used to test for the effect of number of Bd exposures on Bd abundance within the fourth exposure period for the first immunological resistance experiment (see Supplementary Methods for a justification and for additional details).
In our behavioural resistance experiment, we tested for avoidance of Bd using a linear mixed-effects model (package: lme4, function: lmer) with a binomial error distribution. We nested number of Bd exposures within frog and frog within experiment (that is, treated frog and experiment as random effects) and tested whether the proportion of observations on the Bd+ side of the container differed from a 50:50 expectation, allowing us to discriminate between innate and learned avoidance. We tested for main effects of number of Bd exposures (continuous predictor), experiment, deviation from a null 50:50 expectation, and a number-of-Bd-exposures-by-deviation-from-null interaction (see Supplementary Information for additional details). We also conducted post-hoc analyses to determine when frogs significantly avoided Bd, with 0, 1, or 2 Bd infections (with a Bonferroni alpha adjustment; α = 0.016).
Compliance statement
All experiments were approved by the Institutional Animal Care and Use Committee of the University of South Florida.
Extended Data
Extended Data Figure 1. Cumulative survival of Osteopilus septentrionalis.
a, b, Cuban treefrog survival in the first (a) and second immunological resistance experiments (b) with 1, 2, 3 or 4 exposures (previous infections were cleared with heat) to live Batrachochytrium dendrobatidis (Bd). Mortality was greater in the second immunological resistance experiment because we provided six weeks for Bd to grow on the frogs after the final Bd exposure, whereas the frogs were cleared of Bd after only 11 days of growth in the first immunological resistance experiment so that we had ample frog survival for subsequent immunological analyses. Naivety was based on the state of the frog before Bd exposure during the fourth exposure period; thus frogs exposed to Bd for the first time during the fourth exposure period were classified as naive because they had not previously been exposed to Bd.
Extended Data Figure 2. Effects of 1–4 exposures to Batrachochytrium dendrobatidis (Bd) on Bd abundance on frogs (Osteopilus septentrionalis; not standardized by weight).
a, b, Effects of exposures on mean Bd abundance (zoospore genome equivalents (GE) ± s.e.m.) after exposure period 3 in the first immunological resistance experiment (a) and exposure period 4 in the second immunological resistance experiment (live Bd exposures: circles and solid line; dead Bd exposures: squares and dotted line) (b). The best-fit lines are based on predicted values from the implemented zero-inflated negative binomial. Naivety was based on the state of the frog before Bd exposure during the focal exposure period (third or fourth exposure period depending on what is being displayed). Thus, frogs exposed to Bd for the first time during the focal exposure period were classified as naive because they had not previously been exposed to Bd.
Extended Data Figure 3. Effects of 0–4 exposures to live Batrachochytrium dendrobatidis (Bd) on the abundance and efficacy of skin peptides extracted from frogs (Osteopilus septentrionalis).
a, Mean (± s.e.) skin peptide abundance (n = 8, 9, 17, 17, and 18 for 0–4 Bd exposures, respectively). b, Mean (± 95% confidence interval) efficacy of skin peptides at inhibiting Bd, measured as the difference between Bd growth rate in the presence and absence of a standardized concentration of skin peptides (n = 8, 8, 10, 11, and 10 for 0–4 Bd exposures, respectively). See Methods for details on how Bd was quantified and growth rates were calculated. Naivety was based on the state of the frog before Bd exposure during the fourth exposure period; thus frogs exposed to Bd for the first time during the fourth exposure period were classified as naive because they had not previously been exposed to Bd.
Extended Data Figure 4. Cumulative survival of Osteopilus septentrionalis in the second immunological resistance experiment with 1, 2, 3 or 4 exposures to dead Batrachochytrium dendrobatidis (Bd) followed by an exposure to live Bd.
Naivety was based on the state of the frog before Bd exposure during the fourth exposure period; thus frogs exposed to Bd for the first time during the fourth exposure period were classified as naive because they had not previously been exposed to Bd.
Extended Data Table 1.
Description of when exposure to live or dead Batrachochytrium dendrobatidis (Bd) occurred (designated by x) during the first and second immunological resistance experiments
|
Bd exposure-clearance period* |
||||
|---|---|---|---|---|
| Total no. of exposures to live or dead Bd | 1 | 2 | 3† | 4†‡ |
| 0§ | ||||
| 1§ | X | |||
| 2∥ | X | X | ||
| 3 | X | X | X | |
| 4 | X | X | X | X |
All frogs, regardless of treatment, were exposed to the same temperature regimes.
Statistical analyses only occurred within the third or fourth exposure-clearance periods rather than across exposure-clearance periods.
For the first immunological resistance experiment, immunological assays occurred after the fourth exposure-clearance period.
Frogs in this treatment were Bd-naive throughout the experiment.
Frogs in this treatment were Bd-naive during the third but not the fourth exposure-clearance period.
Extended Data Table 2.
Number of frogs swabbed for, and infected with, Batrachochytrium dendrobatidis (Bd) before each experiment began, and clearance temperatures, number of frogs cleared of Bd, and number of Bd clearances (includes multiple clearances per frog) in each experiment
| Experiment | No. of frogs swabbed before experiment | Bd prevalence before experiment (%) | Clearance temperature (°C) | No. of frogs cleared of Bd* | Incidents of Bd clearance (counts multiple clearances per frog)* |
|---|---|---|---|---|---|
| First Behavioral Resistance | 20 | 0 | 30 | 30 | 30 |
| Second Behavioral Resistance | 20 | 0 | 30 | 30 | 60 |
| First Immunological Resistance | 20 | 0 | 32 | 71 | 172† |
| Second Immunological Resistance | 20 | 0 | 30 | 80 | 117† |
Heat-induced clearance of Bd infections was 100% effective.
The difference in Bd clearance incidents between the first and second immunological resistance experiments was because all surviving frogs were cleared of their infections at the end of the first immunological resistance experiment before being shipped to Vanderbilt University for immunological assays and because of minor differences in survival between experiments.
Extended Data Table 3.
Sample size, Batrachochytrium dendrobatidis (Bd) prevalence, and mortality from each experiment
| Experiment | Species tested | Treatment* | n | Prevalence (%)† | No. of frogs that died‡ | Mortality (%) |
|---|---|---|---|---|---|---|
| First Behavioral Resistance | Bufo quercicus | 0 exposures to live Bd (naïve)§ | 10 | - | 0 | 0 |
| First Behavioral Resistance | Bufo quercicus | 1 exposure to live Bd (naïve)§∥ | 10 | 100 | 0 | 0 |
| Second Behavioral Resistance | Bufo quercicus | 0 exposures to live Bd (naïve)§ | 10 | - | 0 | 0 |
| Second Behavioral Resistance | Bufo quercicus | 1 exposure to live Bd (naïve)§∥ | 10 | 100 | 0 | 0 |
| Second Behavioral Resistance | Bufo quercicus | 2 exposures to live Bd§∥ | 10 | 100 | 0 | 0 |
| First Immunological Resistance | Osteopilus septentrionalis | 0 exposures to live Bd (naïve)§ | 10 | - | 1 | 10 |
| First Immunological Resistance | Osteopilus septentrionalis | 1 exposure to live Bd (naïve)§ | 10 | 100 | 3 | 30 |
| First Immunological Resistance | Osteopilus septentrionalis | 2 exposures to live Bd§∥ | 20 | 100 | 3 | 15 |
| First Immunological Resistance | Osteopilus septentrionalis | 3 exposures to live Bd§∥ | 20 | 100 | 2 | 10 |
| First Immunological Resistance | Osteopilus septentrionalis | 4 exposures to live Bd§∥ | 20 | 95 | 1 | 5 |
| Second Immunological Resistance | Osteopilus septentrionalis | 0 exposures to live Bd (naïve)§ | 20 | - | 0 | 0 |
| Second Immunological Resistance | Osteopilus septentrionalis | 1 exposure to live Bd (naïve)§ | 20 | 95 | 1 | 5 |
| Second Immunological Resistance | Osteopilus septentrionalis | 2 exposures to live Bd§∥ | 20 | 77 | 2 | 10 |
| Second Immunological Resistance | Osteopilus septentrionalis | 3 exposures to live Bd§∥ | 20 | 85 | 0 | 0 |
| Second Immunological Resistance | Osteopilus septentrionalis | 4 exposures to live Bd§∥ | 20 | 70 | 0 | 0 |
| Second Immunological Resistance | Osteopilus septentrionalis | 0 exposures to dead Bd (naïve)§¶ | 20 | - | 0 | 0 |
| Second Immunological Resistance | Osteopilus septentrionalis | 1 exposure to dead Bd (naïve)§¶ | 20 | 100 | 3 | 15 |
| Second Immunological Resistance | Osteopilus septentrionalis | 2 exposures to dead Bd§¶ | 20 | 94 | 2 | 10 |
| Second Immunological Resistance | Osteopilus septentrionalis | 3 exposures to dead Bd§¶ | 20 | 71 | 1 | 5 |
| Second Immunological Resistance | Osteopilus septentrionalis | 4 exposures to dead Bd§¶ | 20 | 71 | 3 | 15 |
| Cashins et al. 2013# | Litoria booroolongensis | 1 exposure to live Bd, no exposure to itraconazole (naïve) | 28 | 50 | 4 | 14.3 |
| Cashins et al. 2013# | Litoria booroolongensis | 1 exposure to live Bd, after previous exposure to itraconazole (naïve) | 11 | 91 | 2 | 18.2 |
| Cashins et al. 2013# | Litoria booroolongensis | 2 exposures to live Bd, first exposure cleared with itraconazole | 32 | 63 | 5 | 15.6 |
Immunological defences of frogs were naïve to Bd upon the first exposure because they had not previously been exposed to Bd. Do not confuse the treatments in this column for the behavioural resistance experiments with the x-axis label on Fig. 1, which emphasizes number of previous not present exposures to Bd.
Values are for prevalence at the end of the behavioural resistance and Cashin et al. experiments, at the end of exposure period 3 for the first immunological resistance experiment, and at the end of exposure period 4 for the second immunological resistance experiment.
Cumulative number of frogs that died up to the final swabbing period, with the exception of the second immunological resistance experiment, where data represent cumulative number of frogs that died up to 11 days after the final Bd exposure to match the growth periods in the behavioural and first immunological resistance experiments.
Frogs in these treatments were exposed to exactly the same temperature conditions regardless of whether they were exposed to live Bd or not.
Frogs exposed to live Bd more than once had their previous infections cleared using heat exposures.
Frogs in these treatments were challenged with live Bd at the end of the experiment to evaluate how previous exposures to dead Bd affected actual Bd growth on the frogs.
Ref. 20.
Supplementary Material
Acknowledgements
We thank W. Holden and J. Pask for their assistance with the immunological assays, J. Cohen for comments on the manuscript, and our undergraduate assistants H. Folse, S. Lopez, L. Soto, S. Hekkanen and E. Creasey. We also thank V. Vasquez and J. Longcore for providing the Bd isolates used in these experiments. Funds were provided by grants from the National Science Foundation (DEB 0516227 and EF-1241889 to J.R.R. and IOS-1121758 to L.A.R.-S.), the National Institutes of Health (R01GM109499 to J.R.R.), the US Department of Agriculture (NRI 2006-01370 and 2009-35102-0543 to J.R.R.), the US Environmental Protection Agency grant (STAR R83-3835 and CAREER 83518801 to J.R.R.), and the NSF RCN “Refining and Diversifying Ecoimmunology”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Footnotes
Author Contributions T.A.M., T.R.R., J.R.R., B.F.S., N.T.H. and J.M.B. conceived and designed the first immunological resistance experiment, T.A.M., J.R.R., B.F.S., S.M.B., N.T.H., N.O. and J.M.B conceived and designed the second immunological resistance experiment, M.D.V. and J.R.R. conceived and designed the behavioural resistance experiment. T.A.M. directed the first and second immunological resistance experiments and M.D.V. directed the behavioural resistance experiments. T.A.M., B.F.S., S.M.B., N.T.H., J.M.B., G.L., N.T., S.Y. and M.D.V. conducted the first and second immunological resistance experiments; M.D.V. and K.D. conducted the behavioural resistance experiments, and T.A.M., L.K.R., J.S.F. and L.A.R.-S. conducted the antimicrobial peptide collection and lymphocyte assays. T.A.M. and J.R.R. conducted the statistical analyses for the first and second immunological resistance experiments and D.J.C. consulted on these analyses; M.D.V. and J.R.R. conducted the statistical analyses for the behavioural resistance experiment. J.R.R. wrote the manuscript and handled all submissions and revisions. T.A.M. and B.F.S. wrote parts of the methods, and all authors contributed to its editing.
The authors declare no competing financial interests.
Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.
Supplementary Information is available in the online version of the paper.
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