Rhinella marina chemical cue interactions

TOAD BREEDING
Adult cane toads were collected by hand from sites in Western Australia (WA: Kununurra – 15.778477, 128.744960; Doongan – 15.390413, 126.293106) and the Northern Territory (NT: Middle Point – 12.579564, 131.313918; Mary River Park – 12.904422, 131.651241) and brought back to our laboratory at Middle Point. Pairs of adult toads from each locality were injected with leuprorelin acetate (Lucrin, Abbott Australasia) to induce breeding (see Hayes et al., 2009 for detailed methods). Newly laid clutches of eggs were kept in individual 18 L tubs in unchlorinated bore water at room temperature (25 °C) and aerated until they reached the stages required for our experimental trials.

EFFECTS OF EXPOSURE TO THE SUPPRESSION CUE
Suppression treatment
Plastic aquaria (1 L, 120mm x 170mm x 68mm) each holding 10 hatchlings (Gosner stage 18, not yet free swimming, Gosner, 1960) in 750 mL of bore water, were assigned to either a ‘suppression’ or ‘control’ treatment. We placed a flyscreen mesh container (50mm x 70mm x 20mm, holes 1 mm x 1 mm) into the water in each aquarium, and added three live cane toad tadpoles (Gosner stage 29–33, previously acquired from an older Middle Point clutch) to the container for each of the ‘suppression’ aquaria. In ‘control’ aquaria the mesh container remained empty. The mesh containers were left in place for approximately 48 h, until the hatchlings reached Gosner stage 25 (i.e. had developed into free-swimming tadpoles). Five of those free-swimming tadpoles were randomly selected from each aquarium, and transferred into new aquaria with fresh bore water. They were fed with crushed algae wafers each day (to excess), and we changed the water every second day. These tadpoles were the source of ‘suppression’ and ‘control’ treatment tadpoles for the experiments detailed below. Each tadpole was used in only one of the following three types of trials.

Trapping rates
Ten days post-exposure, 20 tadpoles from each treatment (from a mixture of holding aquaria) were randomly allocated to trays of water (680 x 420 x 70 mm), and left to acclimate for one hour. These trays were assigned to either ‘toxin trapping’ or ‘no toxin trapping’ (control), creating the following four treatments: ‘suppressed + toxin trap’, ‘suppressed + control’, ‘not suppressed + toxin trap’ and ‘not suppressed + control’ (for 3 clutches, with 3–5 replicates each). Tadpole traps were transparent 1 L plastic containers (120mm x 170mm x 68mm) with one wall containing an inward-pointing funnel (funnel: length 35 mm, largest diameter 44 mm, smallest diameter 12 mm), with one trap per tray. Traps were placed against the middle of the smaller wall of the tray, with the opening of the funnel facing into the tray. Toxin baits (attractant cues) were made by squeezing 200 mg of fresh toxin from the parotoid glands of adult female toads into a cup filled with 30 mL of bore water (on the advice of Crossland et al., 2012). The bait was left to sit for half an hour at room temperature before being added to the trap in the ‘toxin trapping’ treatments; an equivalent volume of bore water was simultaneously added to the control traps. The number of tadpoles inside each trap was counted after 90 min.
We also repeated these trials as a separate experiment (using different tadpoles, suppressed and not suppressed), using food (0.5 g of algae wafers) as a bait to determine whether patterns observed in the above trials were seen in response to cues from other types of food as well as in response to the conspecific attractant cue.

Response to alarm cues
Twelve days post-exposure, 20 tadpoles from each suppression treatment were placed into individual 1 L plastic aquaria (120mm x 170mm x 68mm) filled with 250 mL of bore water, and left to acclimate for 1 h. Aquaria were assigned to either ‘alarm cue’ or ‘water cue’ (control), such that there was a total of four treatments; ‘suppressed + alarm cue’, ‘suppressed + control’, ‘not suppressed + alarm cue’ and ‘not suppressed + control’ (3 clutches, 10 replicates of each treatment). The alarm cue was prepared by rapidly macerating 3 g of live conspecific tadpoles, combined with 20 mL of bore water, and filtering the mixture through a nylon mesh net (holes 200 μm). A drop of food colouring was added to enable us to see the alarm cue dispersing through water. The control cue consisted of 20 mL of bore water with a drop of food colouring. A syringe was used to inject 1 mL of either the alarm or control cue into the water along the edge of each aquaria. The tadpole in the aquaria was encouraged to swim towards the cue by gently touching its tail. The tadpole’s reaction as it encountered the cue was scored as either ‘repulsed’ (rapid u-turn, sudden onset of immobility and dropping to the bottom of the aquaria, or suddenly swimming rapidly around the perimeter of the aquaria) or ‘not repulsed’ (tadpoles continued to behave as they had been before encountering the cue). Each tadpole was encouraged to encounter the cue three times, and the proportion of times it was ‘repulsed’ was recorded.

Vulnerability to predation
Two days post-exposure, 10 tadpoles from each suppression treatment were weighed (g) and randomly allocated to plastic tubs (360 x 180 x 200 mm) filled to a depth of 10 cm with bore water (4 clutches, 6–8 replicates per clutch per treatment). Tadpoles were left to acclimate for 1 h. At 0700 h, we added one water bug (Diplonychus sp.) to each tub, along with a floating stick (100 mm long) to enable the bug to rest at the top of the water. The number of tadpoles consumed by the bug was monitored for 24 h. At 10 days post-exposure, these trials were repeated with other tadpoles (same clutches, 4–8 replicates per clutch per treatment).

EFFECTS OF EXPOSURE TO THE ALARM CUE
Alarm treatment
Cane toad hatchlings (3 clutches, Gosner stage 18–19) were placed into 1 L clear plastic aquaria (10 per tub) holding 750 mL of bore water, and left to reach Gosner stage 25. Five viable tadpoles from each aquarium were then randomly selected and placed into a new aquarium. Half of these aquaria were allocated to the ‘alarm’ treatment, and half to the ‘control’ treatment. Alarm cue was prepared as above, but without the addition of dye. Each morning for 7 days, we added 1 mL of alarm cue into each of the ‘alarm’ aquaria, and 1 mL of bore water into the ‘control’ aquaria. We fed the tadpoles crushed algae wafers each afternoon, and changed the water every second day.

Trapping rates
Eight days post-exposure, 20 tadpoles from each treatment were randomly assigned to trays of bore water, and left to acclimate for 1 h. These trays were assigned to either ‘toxin trapping’ or ‘no toxin trapping’ (control), such that there was a total of four treatments: ‘alarm + toxin trap’, ‘alarm + control’, ‘control + toxin trap’ and ‘control + control’ (2–5 replicates each). Toxin baits were made and trapping trials carried out as described above.