Experimental infections
To investigate if skin secretions act as a defense mechanism by the host
or as cue for the parasite, or both, we infected toads that had either
intact or reduced skin secretions (Table S1, S2). We used 79
captive-raised toads, comprising 20 individuals (10 from each clutch)
per treatment group: 20 range-core toads that we infected with
range-core L3, 19 range-core toads infected with invasion-front L3, 20
invasion-front toads infected with invasion-front L3, and 20
invasion-front toads infected with range-core L3. Toads were
approximately two months old when we commenced the experiment, and were
housed individually in plastic boxes (15 x 10 x 7 cm). We assigned
individuals to treatment groups such that mean snout-urostyle length
(SUL) was similar among treatment groups (ANOVA: F (6,
72) = 0.30, p = 0.94).
Directly before infection, we reduced skin secretions on half the toads
by bathing the animal in water and then swabbing both the dorsal and
ventral surface 15 times with a cotton bud (Fig. 1). This process was
conducted twice per individual to remove most of the skin secretions
(Walker et al. 2015). Control toads were also bathed and
mock-swabbed to simulate the same handling time and potential stress as
the other group. That is, we swabbed the toad with a cotton bud covered
with a non-absorptive plastic film (parafilm ‘M’, Pechiney Plastic
Packaging, Chicago, USA), rolling the bud over the body but removing few
if any skin secretions (Christian et al. 2021). For infection, we
placed toads individually into round containers (40 mm diameter) lined
with filter paper (Whatman 1, Whatman International Ltd, Maidstone,
England) containing 20 L3 in 300 µl water. We left the toads in the
infection chamber for 18 h, after which they were placed back in their
housing enclosure, and then rinsed the infection chamber with water onto
a clean Petri dish to count the number of remaining L3 using a
microscope (Leica M60). On the 15th day
post-infection, we euthanized the toads by placing them in a tricaine
methanesulfonate bath (Crossland & Shine 2011) and dissected them to
count the number of established nematodes in the lungs. We calculated
the proportion of L3 entering the lung by dividing the number of L3 that
established in the lungs by the number of L3 that entered the host
(Mayer et al. 2021).