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