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).