10.5061/DRYAD.BG79CNP8F
McCann, Samantha
0000-0002-5408-8744
University of Sydney
Crossland, Michael
,
Greenlees, Matthew
,
Shine, Richard
,
Data from: Field trials of chemical suppression of embryonic cane toads
(Rhinella marina) by older conspecifics
Dryad
dataset
2020
Rhinella marina
2021-07-13T00:00:00Z
2021-07-13T00:00:00Z
en
18138 bytes
2
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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 p > .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.