Introduction
Emerging infectious diseases are one of the largest threats to global biodiversity, but despite this, disease is historically one of the least studied issues in conservation (Lawler et al. 2006). Amphibians, for example, are one of the most threatened vertebrate taxon (Stuartet al. 2004) and have experienced extreme global declines because of the pathogenic fungus Batrachochytrium dendrobatidis (Bd). This pathogen is associated with the extinction and extirpation of hundreds of amphibian species globally and remains an imminent and persistent threat to many extant species (Scheele et al. 2019). Additionally, Bd is unlikely to be extirpated given its nearly global distribution and the presence of non-amphibian hosts (McMahon et al. 2013; Brannelly et al. 2015; Scheele et al. 2019).
Bd has a mobile zoospore stage that produces proteolytic enzymes that degrade elastin proteins in the epidermal layer of the host (Symondset al. 2008; Moss et al. 2010), as well as polyamine spermidine, methylthioadenosine, and tryptophan, all of which are immunomodulators (Rollins-Smith et al. 2015; Rollins-Smithet al. 2019). When crayfish were exposed to Bd metabolites in the absence of the fungus, the metabolites induced crayfish mortality via gill damage (McMahon et al. 2013). Additionally, tadpoles exposed to Bd metabolites in the absence of infection had increased developmental speed (Romansic et al. 2011; McMahon et al.2019); tadpoles can utilize developmental plasticity to avoid an unsuitable environment (Newman 1988; Rohr et al. 2004). Although admittedly anthropomorphic, the damage to host tissue caused by the enzymes in the metabolites is likely painful. This could set the stage for learned avoidance of Bd through Classical or Pavlovian Conditioning. An innate response, in this case avoidance of pain, is paired with a second stimulus, cues from Bd, to induce learned avoidance of Bd. There is precedent for this, as we already know amphibians can learn to avoid pathogenic fungi. For example, bullfrog (Rana catesbeiana ) tadpoles use chemical cues to avoid conspecifics infected with the fungus Candida humicola (Kiesecker et al. 1999), and oak toads (Bufo quercicus ) learn to avoid Bd after experiencing just one Bd infection and clearance event (McMahon et al. 2014).
While oak toads can learn to avoid Bd after exposure (McMahon et al. 2014), we do not know how common this learned avoidance response is across amphibian species, nor do we know what specific Bd cues elicit the avoidance response. Here, we tested whether four species of amphibians – Cuban treefrogs (Osteopilus septentrionalis ; family: Hylidae), southern toads (Bufo terrestris ; family: Bufonidae), greenhouse frogs (Eleutherodactylus planirostris ; family: Eleutherodactylidae), and pine woods treefrogs (Hylafemoralis ; family: Hylidae) – exhibited innate or learned avoidance of Bd. We hypothesized that, like oak toads, all four species would learn to avoid Bd after exposure to and clearance of the pathogen. For any species that exhibited avoidance behavior, we then exposed Bd-naïve and Bd-experienced individuals to different components of Bd to determine which cues are used to avoid Bd. Generally, we crossed the presence and absence of Bd metabolites with the presence and absence of live and dead zoospores. Given that metabolites are likely painful because they damage amphibian skin, we predicted that the metabolites would be an important component of Bd that triggered any learned avoidance response. If amphibians learn to avoid Bd without exposure to live infectious propagules, then it would suggest that a vaccine including a non-infectious cue, such as metabolites, might be effective at inducing an acquired behavioral resistance response that could increase the likelihood that amphibians could persist with Bd.
Materials and Methods