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.