DOI: 10.5061/dryad.bg79cnp8f
title: Data from: Field trials of chemical suppression of embryonic cane toads (Rhinella marina) by older conspecifics

description: 1. Laboratory experiments have shown that the viability of embryos of the
 invasive cane toad (Rhinella marina) can be reduced by exposure to
 chemical cues from older conspecific larvae. These effects (very strong in
 laboratory trials) may offer an exciting new approach to controlling this
 problematic invasive species in Australia. However, the degree to which
 the method works in natural environments has yet to be assessed. 2. Our
 experiments in the laboratory and in semi-natural outdoor waterbodies show
 that chemical cues from tadpoles do indeed suppress the growth,
 development and survival of conspecific larvae that are exposed as embryos
 and do so in a dose-dependent manner; higher tadpole densities cause
 greater suppression of embryos. 3. In semi-natural outdoor waterbodies,
 suppressor-exposed tadpoles were less than half as likely to survive to
 metamorphosis as were controls, and were much smaller when they did so and
 hence, less likely to survive the metamorph stage. Additionally, female
 cane toads were less likely to oviposit in a waterbody containing
 free-ranging (but not cage-enclosed) tadpoles, suggesting that the
 presence of tadpoles (rather than the chemical cues they produce) may
 discourage oviposition. 4. Broadly, our results suggest that the
 suppression effect documented in laboratory studies does indeed occur in
 the field also, and hence that we may be able to translate that approach
 to develop new and more effective ways to reduce rates of recruitment of
 peri-urban populations of cane toads in their invasive range.
description: Field trials of cane toad suppression pheromones
 2.1  |  Experimental protocols
 2.1.1  |  Laboratory trials on suppressor density
 Adult cane toads were collected by hand from Middle Point,
 Northern Territory (-12.579602, 131.313863) and brought back to a nearby
 laboratory where they 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 placed in individual
 18 L tubs in unchlorinated water at a constant temperature (26 °C) and
 aerated until they reached developmental stage 18 (Gosner,
 1960). Four circular pools (2200 mm diameter) were
 filled with 1800 L of unchlorinated water. An enclosed mesh net (700 x 400
 x 300 mm, mesh size 1 x 1 mm) was placed into the centre of each pool and
 secured so that the top of the enclosure was 5 cm above the water surface.
 Twelve hours before the experiment began, each net was filled with 300,
 30, 3 or zero (control) live cane toad tadpoles (stage 28–34) from an
 older Middle Point clutch. When the experimental clutch
 reached Gosner stage 18, 10 of the hatchlings were placed in each of 32
 enclosed circular plastic containers (diameter 76 mm, height 24 mm) with
 mesh sides to allow water flow-through. Eight of these containers were
 then placed into each of the four pools, and weighted so that they sat at
 the bottom of the water column. Four of the containers were positioned at
 the edge of the pool (75 cm from the tadpole net, = ‘far’) and four were
 placed beside the net (‘near’). The containers were left in the pools for
 48 h (until hatchlings reached stage 25) to allow exposure to cues from
 the netted tadpoles. They were then removed and five of the now
 free-swimming tadpoles from each experimental container were randomly
 selected and placed together into a clean 1 L plastic container filled
 with 750 mL of fresh unchlorinated water. The tadpoles were fed crushed
 algal wafers daily, and water was changed every second day. Ten days
 later, the tadpoles were weighed and their developmental stages recorded.
 We ran this experiment three times, using three different clutches of
 hatchlings, and exposing them to three different clutches of
 tadpoles. 2.1.2  |  Field and laboratory trials on
 effects of suppression Two clutches of eggs were
 obtained from cane toads collected from Kununurra, and raised as described
 above. When the tadpoles reached stage 18, 10 hatchlings were placed in
 each of eight enclosed circular plastic containers with mesh sides
 (diameter 76 mm, height 24 mm), and this was repeated for each clutch. For
 the field trials, two of these containers (both containing hatchlings from
 the same clutch) were then placed into each of the eight ponds (four ponds
 per clutch) and weighted to sit at the bottom of the water column (20 cm
 deep). Eight replicate ponds (5 x 4 m, 1 m depth at deepest end, gradient
 to 0 m at opposite end) were dug 2 m apart, in a clay-based depression in
 bushland 15 km from Kununurra, Western Australia (-15.827949, 128.856982;
 Figure 1). The ponds were lined with plastic sheeting (100 μm thick) to
 help retain water, and covered with 20 mm of natural sediment and 28 L of
 benthic leaf litter sourced from a nearby waterbody. Ponds were each
 filled with 7500 L of water, sourced from the local Lake Kununurra (from
 an area where toads do not breed), and given 48 h to settle. An enclosed
 mesh net (400 x 300 x 300 mm, mesh size 1 x 1 mm) was placed into each
 pond and secured so that the top of the enclosure was 5 cm above the water
 surface. Twelve hours before the experiment began, half of the nets were
 filled with 30 cane toad tadpoles (Gosner stage 28–34) field-caught from
 two local populations and half of the nets were left empty (controls).
 This suppression treatment equalled a density of 0.004 tadpoles/L, falling
 between the “3 tadpole” (0.002 tadpoles/L) and “30 tadpole” (0.02
 tadpoles/L) treatments in the previous laboratory experiment.
 For the laboratory trials, another 10 hatchlings were placed into
 each of 10 plastic aquaria (1 L) containing 750 mL of unchlorinated water,
 and this was repeated for each clutch. These were assigned to either a
 ‘suppression’ or ‘control’ treatment. We placed a flyscreen mesh enclosure
 (60 x 40 x 30 mm, mesh holes 1 x 1 mm) into each aquarium, and added two
 live cane toad tadpoles (one from each of the wild populations used for
 the pond enclosures) to the container for each of the ‘suppression’
 aquaria. In ‘control’ aquaria the mesh container remained empty. The
 aquaria were kept in the laboratory at 30 °C. The pond
 and laboratory treatments were both left for 48 h (until the hatchlings
 reached stage 25, when they become capable of swimming) to allow exposure
 to tadpole-derived cues, after which they were removed. From each
 container five of the now free-swimming tadpoles were randomly selected
 and placed into a clean 1 L plastic container filled with 750 mL of fresh
 unchlorinated water. The tadpoles were fed crushed algal wafers daily and
 water was changed every second day. Ten days later, tadpoles were weighed
 and their developmental stages recorded. They were then returned to the
 laboratory and raised until they either died or reached metamorphosis. If
 metamorphosis was reached, the days taken to reach metamorphosis and the
 size of the metamorph at emergence were recorded. This
 experiment was repeated with a further two clutches, allowing enough time
 for the previous suppression cues to no longer be present in the ponds
 (Clarke et al. 2015, 2016), and using different suppressor tadpoles.
 Although measurements were taken at 10 days growth for all clutches
 tested, time constraints meant that only the first two clutches were run
 to metamorphosis. 2.1.3  |  Trials of oviposition
 behaviour We erected walls of plastic sheeting (500 mm
 high) across the middle of each of the ponds, to divide each pond evenly
 into two. Each half of the original pond held 2500 L, with no water flow
 between the two sides but a common bank. Twelve hours before the
 experiment began we placed a mesh enclosure (200 x 200 x 150 mm) into the
 middle of each half-pond. One side was randomly allocated to the
 ‘suppression’ treatment and the other side left as a control. In the
 suppression sides, 30 tadpoles (stage 28–34) from a mixture of two wild
 clutches were placed into the mesh enclosure. Fences (600 mm tall) around
 each pond (enclosing both of the half-ponds as a single unit) excluded any
 wild cane toads. Adult cane toads were collected by
 hand from around Kununurra, and were injected with leuprorelin acetate
 (Lucrin, Abbott Australasia) to induce breeding. One female and two males
 were then placed inside the fence of each pond and left overnight. The
 next morning we collected any clutches laid in the ponds and recorded the
 side (half-pond) in which they had been laid. If no eggs had been laid the
 replicate was removed from the data set. This experiment was repeated
 (with fresh adult toads and fresh suppressor tadpoles) until 10 clutches
 had been laid. We then repeated the experiment but
 instead of restraining the suppression tadpoles (‘enclosed suppression’),
 the mesh enclosures were removed and suppression tadpoles were placed
 directly into the pond, allowing them to swim freely (‘non-enclosed
 suppression’). The control side of the pond contained no tadpoles. This
 experiment was repeated with fresh toads and fresh suppressor tadpoles
 until seven clutches had been laid (the work was then terminated because
 of unsuitable weather). 2.2  |  Statistical
 analysis  Data were normally distributed, so we used
 parametric tests. 2.2.1  |  Laboratory trials on
 suppressor density The position of embryos in the pond
 (near or far) had no effect on mass or stage of tadpoles (ANOVA with
 location as factor, all <i>p</i> &gt; .05), and so these
 treatments were combined for the analysis. The mass and stage of the five
 tadpoles raised in each 1 L container were averaged, to create one
 replicate. To test for an effect of suppressor density on the mass and
 stage of tadpoles, we ran two individual ANOVAs in SPSS v21 (IBM, Armonk,
 NY), with density treatment as the factor, and included clutch as a random
 factor. We then ran Tukey’s post-hoc tests to determine which treatments
 contributed to the differences observed.
 2.2.2  |  Field and laboratory trials on effects of
 suppression To test for an effect of suppression
 treatment on survival, mass and stage we ran two individual two-way ANOVAs
 in JMP v9 (SAS Institute, Cary, NC), with ‘Suppression treatment’ and
 ‘Location’ as the factors, and including ‘Clutch’ as a random factor. To
 test for an effect of suppression on the mass of metamorphs and on the
 days taken for individuals to reach metamorphosis, we ran separate ANOVAs
 in JMP with ‘Suppression treatment’ and ‘Location’ as the factors and
 including ‘Clutch’ as a random factor.
 2.2.3  |  Trials of oviposition behaviour To
 test for an effect of enclosed suppression treatment on the likelihood of
 toads laying in a waterbody, we ran a binomial test in SPSS comparing the
 number of times toads laid in the suppression treated side of the pond and
 the number of times they laid in the control side. We repeated this
 analysis to test for an effect of non-enclosed suppression treatment on
 the likelihood of toads laying in a waterbody.