Collection of toads and lungworms
We obtained cane toads from two geographic areas: (1) Mareeba,
Queensland (17.03° S, 145.43° E), close to the original introduction
site (hereafter ‘range-core’, toads present >80 years), and
(2) Halls Creek, Western Australia (-18.23° S, 127.66° E; hereafter
‘invasion-front’, toads present <5 years; Fig. 1). We kept
toads in Middle Point, Northern Territory (-12.56° S, 131.32° E; Fig.
1), individually in 20 L containers. To obtain naive, parasite-free
toads, two pairs of adult toads from each region were induced to spawn
by subcutaneous injection of the gonadotropin-releasing hormone agonist
leuprorelin acetate (Lucrin, Abbott Australasia) (Hudson et al.2016). Although our sample size of parental toads was low for logistical
reasons, low functional genotypic replication is unlikely to have
affected our findings because cane toads have very low genetic diversity
across Australia, with a consequent high similarity in gene expression
among individuals within populations (Rollins et al. 2015;
Selechnik et al. 2019b).
After tadpoles hatched, we raised them in plastic containers (1165 x
1165 x 780 mm; one container per clutch). Emerging metamorphosed toads
were housed in plastic containers (375 x 295 x 195 mm) separated by
clutch prior to the experimental trials. To obtain parasites, we
collected naturally-infected toads from Innisfail, QLD (range-core, n =
61) and Kununurra, WA (close to the invasion-front; toads present ≤10
years, n = 20), because parasites are absent from the invasion-front
(Phillips et al. 2010). The sites where we collected parasites
were about 200 km from the sites where we collected parental toads, to
yield similar spatial differences between collection sites for parasites
and toads at the range-core and invasion-front. We obtained parasites by
collecting toad feces and by sourcing adult Rhabdias from toad
lungs, which we then cultured in Petri dishes in a mix of aged tap water
and toad feces to raise first-stage Rhabdias larvae to the L3
stage (Langford & Janovy Jr 2009; Kelehear et al. 2012).