Discussion
We found that amphibian species had different abilities to learn to avoid Bd. Neither Bd-naïve nor Bd-experienced Cuban treefrogs, greenhouse frogs and pine woods treefrogs avoided Bd. Bd-naïve southern toads exhibited no avoidance of Bd zoospores or metabolites, but after one Bd infection and clearance, they avoided treatments containing Bd metabolites. Oak toads were also capable of learning to avoid Bd (McMahon et al. 2014) and thus this is the second species in Bufonidae capable of acquired behavioral avoidance of Bd. More research is needed to explore whether the ability to learn avoidance behaviors is influenced by phylogeny.
Bd metabolites triggered the associational behavioral avoidance in southern toads, which is not surprising given that many vertebrate species are capable learning to avoid a cue that is accompanied by a painful stimulus (Dunlop et al. 2006). The metabolites contain proteolytic enzymes and other chemicals that degrade host tissue (Mosset al. 2010; McMahon et al. 2013; Rollins-Smith et al. 2015; Rollins-Smith et al. 2019), and thus exposure to metabolites might indeed be painful. Amphibians may be able to detect these chemical metabolites through the chemo- or pain receptors in their skin (Hillyard & Willumsen 2011), which could facilitate the detection and avoidance of external stimuli through epidermal contact (for example see Kiesecker et al. 1999). Pavlovian conditioning predicts that pain inducing chemicals would produce a strong behavioral avoidance response. This is not only consistent with what we found here but is also consistent with amphibian behavioral avoidance of other directly transmitted pathogens (Kiesecker et al. 1999, Zylberberg et al. 2013). In all of these cases, the behavioral response is likely to lower exposure to pain, which may also lower risk of infection.
Interestingly, the other species of amphibians tested did not exhibit avoidance of Bd when Bd naïve or Bd experienced. The high Bd-induced mortality in the pine woods treefrogs was not unusual for species in the family Hylidae (Scheele et al. 2019). Given this high susceptibility to Bd, we would have expected strong selective pressure for innate behavioral avoidance, but none was detected in this species. This is possibly because these populations may not have an evolutionary history with Bd, which would be necessary for selection of avoidance responses.
We did not see high Bd-induced mortality in the greenhouse frogs and Cuban treefrogs. Because these species appear to be somewhat tolerant to Bd, they may not have experienced strong selective pressures to exhibit innate or learned avoidance of the fungus. Selection pressures are a product of variation in both exposure and susceptibility to the fungus (Sears et al. 2015). In addition to different selection pressures for avoidance, there might also be differences among species in their ability to learn. Hence, the combination of variation in selection pressures and learning abilities might explain the observed variation among amphibian species in their learned avoidance responses to Bd. We encourage additional research that screens for the ability to discriminate between innate and learned avoidance of pathogens and that identifies the cues used to induce any avoidance response. This would give researchers a better understanding of the strength and importance of behavioral avoidance in the field.
Given that human-assisted migration is believed to have introduced Bd to new locations around the planet in the last 150 years (for example, Ouellet et al. 2005; Padgett-Flohr & Hopkins 2009; Tallleyet al. 2015), we suspect that many hosts might not have had an evolutionary history with Bd for avoidance behaviors to have become fixed as innate traits. Consequently, we encourage additional research that discriminates between innate and learned avoidance of pathogens and that identifies the cues used to induce any avoidance response. Moreover, there is a need to better understand the strength and importance of Bd avoidance in the field and the degree of species-level variation in this avoidance.
This research has important implications for the conservation and management of amphibian populations threatened by Bd. The IUCN Amphibian Ark network and other conservation initiatives rescued hundreds of amphibian species threatened by Bd and are maintaining these species in captivity in the hopes that they might be released back into the wild (Venesky et al. 2012; Venesky et al. 2013). Reintroduction attempts have often failed presumably because of the persistence of Bd in the environment on tolerant hosts (Venesky et al. 2012; McMahon et al. 2013; Venesky et al. 2013). Previous work has suggested that immunization with dead Bd might induce acquired immunological resistance that could facilitate successful reintroductions of captively bred amphibians (McMahon et al.2014). Given that some amphibians can also learn to avoid Bd without exposure to live infectious propagules, our work opens the door to using non-infectious cues to induce an acquired behavioral resistance response in captively bred and perhaps even wild amphibians. Acquired behavioral resistance to Bd could increase the likelihood that certain species can persist in nature with Bd. For this to be successful, a better understanding of which species of amphibians are capable of acquiring immunological or behavioral resistance to Bd is needed. We hope that induction of acquired immunological and behavioral resistance using non-infectious cues of Bd, like Bd metabolites, can reduce Bd-induced amphibian declines and can facilitate the successful re-establishment of captively bred amphibians into the wild.