Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)?

Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either 'cannibal cues' from older conspecifics, or 'alarm cues' from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific-derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.

Mechanisms, costs, and carry-over effects of cannibal-induced developmental plasticity in invasive cane toads.

Inducible defences can improve survival in variable environments by allowing individuals to produce defences if they detect predators. These defences are often expressed as inter-related developmental, morphological, and behavioural changes. However, producing defences can incur costs, which may be expressed immediately and/or during subsequent life stages. In Australia, waterborne cues of potentially cannibalistic conspecific tadpoles induce hatchlings of invasive cane toads to accelerate their developmental rate, thereby reducing their window of vulnerability. However, the mechanisms and costs of such accelerated development are poorly understood, and whether cane toad embryos show cannibal-induced plasticity in other traits is unknown. Here, we found no evidence of altered time of hatching for embryos exposed to non-feeding conspecific cannibal tadpole cues. Additionally, hatchling dispersal behaviours were not affected by exposure to these cues. However, developmental acceleration of hatchlings induced by exposure to tadpole cues was accompanied by reduced hatchling growth, indicating a trade-off between these processes. At the conclusion of the hatchling stage, cannibal-exposed individuals were smaller and morphologically distinct from control siblings. This size reduction affected performance during the subsequent tadpole stage: smaller cannibal-exposed individuals were more likely to die, and initial size tended to be positively associated with subsequent tadpole growth and development across treatments (respectively, p = .07 and p = .06). However, even accounting for variation in initial size, there was an additional negative effect of cannibal exposure on tadpole growth and development, demonstrating that the fitness costs associated with developmental acceleration are not entirely attributable to size reductions.

A biological invasion reduces rates of cannibalism by Japanese toad tadpoles.

Biological invasions can favour rapid changes in intraspecific competitive mechanisms such as cannibalism by imposing novel evolutionary pressures. For example, cane toad (Rhinella marina) tadpoles are highly cannibalistic on eggs and hatchlings in their invasive range in Australia, but not in their native range in South America. Whether such changes in cannibalism occur in invasive populations of other amphibian species is unknown. To explore this question, we collected wild-laid egg clutches of Japanese common toads (Bufo japonicus) from native and invasive populations in Japan, and conducted laboratory experiments to examine cannibalism responses. Contrary to the Australian system, we found that invasion has been accompanied by reduced cannibalistic tendency of B. japonicus tadpoles. This reduction has occurred despite invasive-range B. japonicus eggs/hatchlings being more vulnerable than native-range B. japonicus eggs/hatchlings to cannibalism by native-range conspecific tadpoles, and to predation by native-range frog tadpoles. Our findings thus support the idea that biological invasions can generate rapid changes in rates of cannibalism, but also show that decreases as well as increases can occur. Future work could investigate the proximate cues and selective forces responsible for this rapid decrease in rates of cannibalism by tadpoles in an invasive B. japonicus population.

Choosy cannibals: Targeted consumption of conspecific hatchlings by larval cane toads is triggered by species-specific defensive toxins.

In many species, cannibalism is uncommon and involves nonselective consumption of conspecifics as well as heterospecifics. However, within their invasive Australian range, cane toad larvae (Rhinella marina) specifically target and voraciously consume the eggs and hatchlings of conspecifics, often extirpating entire clutches. In contrast, toad larvae rarely consume the eggs and hatchlings of native frogs. Here, we use laboratory studies to demonstrate that this selective consumption is triggered by species-specific chemical cues: maternally-invested bufadienolide toxins that otherwise defend cane toad eggs and hatchlings against predators. We find that these cues stimulate feeding behaviors in toad tadpoles, such that the addition of bufadienolide toxins to the water column increases predation on eggs, not only of conspecifics, but also of native anuran species that are otherwise usually ignored. In contrast, we find that cannibalism rates on conspecific hatchlings are high and unaffected by the addition of bufadienolide cues. The maternally-invested toxins present in conspecific eggs may therefore be more easily detected post-hatching, at which point tadpole feeding behaviors are induced whether or not additional toxin cues are present. As bufadienolide cues have previously been found to attract toad tadpoles to vulnerable hatchlings, our present findings demonstrate that the same toxin cues that attract cannibalistic tadpoles also induce them to feed, thereby facilitating cannibalism through multiple behavioral effects. Because native fauna do not produce bufadienolide toxins, the species specificity of these chemical cues in the Australian landscape may have facilitated the evolution of targeted (species-specific) cannibalism in invasive cane toad populations. Thus, these bufadienolide toxins confer cost (increased vulnerability to cannibalism in early life-stages) as well as benefit (reduced vulnerability to predation by other taxa).

Competition between the tadpoles of Japanese toads versus frogs.

Competition within and among species can play a key role in structuring the assemblages of anuran tadpoles. Previous studies have reported that tadpoles of the invasive cane toad (Rhinella marina) are more strongly disadvantaged by the presence of native frog tadpoles than by the same number of conspecific toad tadpoles. That effect might arise from a lack of coevolution of the invasive toad with its competitors; and/or from a generalized superiority of frog tadpoles over toad tadpoles. To clarify those possibilities, we conducted experimental trials using the larvae of a native rather than invasive toad (Bufo japonicus formosus in Japan) exposed to larvae of native anurans (the sympatric frogs Rana japonica and Rana ornativentris and the parapatric toad Bufo japonicus japonicus). In intraspecific competition trials, higher densities of B. j. formosus prolonged the larval period and reduced size at metamorphosis, but did not affect survival. In interspecific competition trials, the effects of the other anuran species on B. j. formosus were similar to the effects of the same number of conspecific larvae. This similarity in impact of interspecific versus intraspecific competition argues against any overall competitive superiority of frog larvae over toad larvae. Instead, the vulnerability of larval cane toads to frog tadpoles may result from a lack of coevolutionary history.

The evolution of targeted cannibalism and cannibal-induced defenses in invasive populations of cane toads.

Biotic conflict can create evolutionary arms races, in which innovation in one group increases selective pressure on another, such that organisms must constantly adapt to maintain the same level of fitness. In some cases, this process is driven by conflict among members of the same species. Intraspecific conflict can be an especially important selective force in high-density invasive populations, which may favor the evolution of strategies for outcompeting or eliminating conspecifics. Cannibalism is one such strategy; by killing and consuming their intraspecific competitors, cannibals enhance their own performance. Cannibalistic behaviors may therefore be favored in invasive populations. Here, we show that cane toad tadpoles (Rhinella marina) from invasive Australian populations have evolved an increased propensity to cannibalize younger conspecifics as well as a unique adaptation to cannibalism-a strong attraction to vulnerable hatchlings-that is absent in the native range. In response, vulnerable conspecifics from invasive populations have evolved both stronger constitutive defenses and greater cannibal-induced plastic responses than their native range counterparts (i.e., rapid prefeeding development and inducible developmental acceleration). These inducible defenses are costly, incurring performance reductions during the subsequent life stage, explaining why plasticity is limited in native populations where hatchlings are not targeted by cannibalistic tadpoles. These results demonstrate the importance of intraspecific conflict in driving rapid evolution, highlight how plasticity can facilitate adaptation following shifts in selective pressure, and show that evolutionary processes can produce mechanisms that regulate invasive populations.

Chemical cues that attract cannibalistic cane toad (Rhinella marina) larvae to vulnerable embryos.

Chemical cues produced by late-stage embryos of the cane toad (Rhinella marina) attract older conspecific larvae, which are highly cannibalistic and can consume an entire clutch. To clarify the molecular basis of this attraction response, we presented captive tadpoles with components present in toad eggs. As previously reported, attractivity arises from the distinctive toxins (bufadienolides) produced by cane toads, with some toxins (e.g., bufagenins) much stronger attractants than others (e.g., bufotoxins). Extracts of frozen toad parotoid glands (rich in bufagenins) were more attractive than were fresh MeOH extracts of the parotoid secretion (rich in bufotoxins), and purified marinobufagin was more effective than marinobufotoxin. Cardenolide aglycones (e.g., digitoxigenin) were active attractors, whereas C-3 glycosides (e.g., digoxin, oubain) were far less effective. A structure-activity relationship study revealed that tadpole attractant potency strongly correlated with Na+/K+ ATPase inhibitory activity, suggesting that tadpoles monitor and rapidly react to perturbations to Na+/K+ ATPase activity.

Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader.

In response to novel environments, invasive populations often evolve rapidly. Standing genetic variation is an important predictor of evolutionary response but epigenetic variation may also play a role. Here, we use an iconic invader, the cane toad (Rhinella marina), to investigate how manipulating epigenetic status affects phenotypic traits. We collected wild toads from across Australia, bred them, and experimentally manipulated DNA methylation of the subsequent two generations (G1, G2) through exposure to the DNA methylation inhibitor zebularine and/or conspecific tadpole alarm cues. Direct exposure to alarm cues (an indicator of predation risk) increased the potency of G2 tadpole chemical cues, but this was accompanied by reductions in survival. Exposure to alarm cues during G1 also increased the potency of G2 tadpole cues, indicating intergenerational plasticity in this inducible defence. In addition, the negative effects of alarm cues on tadpole viability (i.e. the costs of producing the inducible defence) were minimized in the second generation. Exposure to zebularine during G1 induced similar intergenerational effects, suggesting a role for alteration in DNA methylation. Accordingly, we identified intergenerational shifts in DNA methylation at some loci in response to alarm cue exposure. Substantial demethylation occurred within the sodium channel epithelial 1 subunit gamma gene (SCNN1G) in alarm cue exposed individuals and their offspring. This gene is a key to the regulation of sodium in epithelial cells and may help to maintain the protective epidermal barrier. These data suggest that early life experiences of tadpoles induce intergenerational effects through epigenetic mechanisms, which enhance larval fitness. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Do Epigenetic Changes Drive Corticosterone Responses to Alarm Cues in Larvae of an Invasive Amphibian?

The developmental environment can exert powerful effects on animal phenotype. Recently, epigenetic modifications have emerged as one mechanism that can modulate developmentally plastic responses to environmental variability. For example, the DNA methylation profile at promoters of hormone receptor genes can affect their expression and patterns of hormone release. Across taxonomic groups, epigenetic alterations have been linked to changes in glucocorticoid (GC) physiology. GCs are metabolic hormones that influence growth, development, transitions between life-history stages, and thus fitness. To date, relatively few studies have examined epigenetic effects on phenotypic traits in wild animals, especially in amphibians. Here, we examined the effects of exposure to predation threat (alarm cues) and experimentally manipulated DNA methylation on corticosterone (CORT) levels in tadpoles and metamorphs of the invasive cane toad (Rhinella marina). We included offspring of toads sampled from populations across the species' Australian range. In these animals, exposure to chemical cues from injured conspecifics induces shifts in developmental trajectories, putatively as an adaptive response that lessens vulnerability to predation. We exposed tadpoles to these alarm cues, and measured changes in DNA methylation and CORT levels, both of which are mechanisms that have been implicated in the control of phenotypically plastic responses in tadpoles. To test the idea that DNA methylation drives shifts in GC physiology, we also experimentally manipulated methylation levels with the drug zebularine. We found differentially methylated regions (DMRs) between control tadpoles and their full-siblings exposed to alarm cues, zebularine, or both treatments. However, the effects of these manipulations on methylation patterns were weaker than clutch (e.g., genetic, maternal, etc.) effects. CORT levels were higher in larval cane toads exposed to alarm cues and zebularine. We found little evidence of changes in DNA methylation across the GC receptor gene (NR3C1) promoter region in response to alarm cue or zebularine exposure. In both alarm cue and zebularine-exposed individuals, we found differentially methylated DNA in the suppressor of cytokine signaling 3 gene (SOCS3), which may be involved in predator avoidance behavior. In total, our data reveal that alarm cues have significant impacts on tadpole physiology, but show only weak links between DNA methylation and CORT levels. We also identify genes containing DMRs in tadpoles exposed to alarm cues and zebularine, particularly in range-edge populations, that warrant further investigation.

The Effects of Conspecific Alarm Cues on Larval Cane Toads (Rhinella marina).

Many aquatic organisms detect and avoid damage-released cues from conspecifics, but the chemical basis of such responses, and the effects of prolonged exposure to such cues, remain poorly understood. Injured tadpoles of the cane toad (Rhinella marina) produce chemical cues that induce avoidance by conspecific tadpoles; and chronic exposure to those cues decreases rates of tadpole survival and growth, and reduces body size at metamorphosis. Such effects suggest that we might be able to use the cane toads' alarm cue for biocontrol of invasive populations in Australia. In the present study, we examined behavioral and ecological effects of compounds that are present in cane toad tadpoles and thus, might trigger avoidance of crushed conspecifics. Four chemicals (L-Arg, L-Leu-L-Leu-OH, L-Leu-L-Ile-OH and suberic acid) induced behavioral avoidance in toad tadpoles at some (but not all) dosage levels, so we then exposed toad larvae to these chemicals over the entire period of larval development. Larval survival and size at metamorphosis were decreased by chronic exposure to crushed conspecifics (consistent with earlier studies), but not by exposure to any of the four chemicals. Indeed, L-Arg increased body size at metamorphosis. We conclude that the behavioral response to crushed conspecifics by cane toad tadpoles can be elicited by a variety of chemical cues, but that consistent exposure to these individual chemical cues does not affect tadpole viability or developmental trajectory. The optimal behavioral tactic of a tadpole may be to flee if it encounters even a single chemical cue likely to have come from an injured conspecific (indicative of predation risk), whereas the continuing presence of that single chemical (but no others) provides a less reliable signal of predation risk. Our data are consistent with results from studies on fish, that suggest a role for multiple chemicals in initiating alarm responses to damage-released cues.

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader.

Background: Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Findings: Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. Conclusions: This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers.

Methods for invasive species control are transferable across invaded areas.

Cane Toads (Rhinella marina) are invasive pests in many parts of the world, including the Japanese island of Ishigaki. Extensive research in Australia has identified promising new methods for control, but also has shown that toads exhibit geographic variation in many traits (suggesting that methods developed in one location may not work in another). Can the approaches developed in Australia play a useful role for controlling this invasive species in Japan? Our experimental trials on Ishigaki Island suggest that these new methods can be successfully applied to Japan. First, Cane Toad embryos exposed to chemical cues of conspecific tadpoles exhibited a reduction in viability (subsequent growth and development). This response appears to be species-specific, with native frog embryos not being affected by exposure to cues from toad tadpoles, and Cane Toad embryos not being affected by exposure to cues from native frog tadpoles. Second, Cane Toad tadpoles were attracted to traps containing water from conspecific eggs, and toxin from adult conspecifics. Third, adult Cane Toads were attracted to acoustic cues of calling males, with sex differences in rates of attraction to specific versions of a synthetic call (males were attracted to choruses whereas females were attracted to low-frequency calls). Our results suggest that the methods developed by Australian researchers are applicable to controlling invasive Cane Toads in Japan.

Biotic Resistance to an Alien Amphibian: Larval Competition between Japanese Frogs and Invasive Cane Toads.

Understanding negative effects of native species on introduced taxa may suggest novel ways to control the invasive species by enhancing such effects. Previous studies have reported that the larvae of invasive cane toads (Rhinella marina) are suppressed by competition with the larvae of native anurans in Australia, but not in North America. We conducted laboratory trials to measure the effect of exposure to the larvae of Japanese frogs (Microhyla ornata, Fejervarya sakishimensis, Rhacophorus owstoni) on rates of survival, growth and development of cane toad tadpoles in Ishigaki Island, in southern Japan. Survival rates were not affected by native species, but competition with Dicroglossids and Rhacophorids (but not Microhylids) strongly reduced rates of growth and development in the tadpoles of cane toads. Dicroglossid tadpoles also reduced the body condition to toad tadpoles in addition to effects on SVL and mass. Encouraging populations of native frogs in toad-invaded areas of Japan thus may help to reduce the numbers of invasive cane toads.

Can we control the invasive cane toad using chemicals that have evolved under intraspecific competition?

Many invasive species experience intense intraspecific competition, because they are abundant in anthropogenically disturbed habitats where few native species persist. Species-specific competitive mechanisms that evolve in this context may offer novel, highly targeted means to control invasive taxa. We conducted laboratory experiments to evaluate the feasibility of this method of control, based on waterborne cues that are produced by tadpoles of the cane toad (Rhinella marina) to suppress the development of conspecific embryos. Our trials examined the nature and species-specificity of the effect, the robustness of the cue to freezing and storage, and the amounts required to suppress toad embryos. Our results were encouraging. The cue appears to be chemical rather than a biological organism, and may well be species-specific; the four species of native anurans that we tested were not influenced by toad larval cues. The cue retains its effectiveness after being frozen, but not after being dried, or after 7 d in water. It is effective at very low concentrations (the amount produced by three tadpoles within 750 L of water). Overall, the cane toad's suppressor pheromone may offer an effective new way to control invasive toads.

The interacting effects of ungulate hoofprints and predatory native ants on metamorph cane toads in tropical Australia.

Many invasive species exploit the disturbed habitats created by human activities. Understanding the effects of habitat disturbance on invasion success, and how disturbance interacts with other factors (such as biotic resistance to the invaders from the native fauna) may suggest new ways to reduce invader viability. In tropical Australia, commercial livestock production can facilitate invasion by the cane toad (Rhinella marina), because hoofprints left by cattle and horses around waterbody margins provide distinctive (cool, moist) microhabitats; nevertheless the same microhabitat can inhibit the success of cane toads by increasing the risks of predation or drowning. Metamorph cane toads actively select hoofprints as retreat-sites to escape dangerous thermal and hydric conditions in the surrounding landscape. However, hoofprint geometry is important: in hoofprints with steep sides the young toads are more likely to be attacked by predatory ants (Iridomyrmex reburrus) and are more likely to drown following heavy rain. Thus, anthropogenic changes to the landscape interact with predation by native taxa to affect the ability of cane toads in this vulnerable life-history stage to thrive in the harsh abiotic conditions of tropical Australia.

Larger body size at metamorphosis enhances survival, growth and performance of young cane toads (Rhinella marina).

Body size at metamorphosis is a key trait in species (such as many anurans) with biphasic life-histories. Experimental studies have shown that metamorph size is highly plastic, depending upon larval density and environmental conditions (e.g. temperature, food supply, water quality, chemical cues from conspecifics, predators and competitors). To test the hypothesis that this developmental plasticity is adaptive, or to determine if inducing plasticity can be used to control an invasive species, we need to know whether or not a metamorphosing anuran's body size influences its subsequent viability. For logistical reasons, there are few data on this topic under field conditions. We studied cane toads (Rhinella marina) within their invasive Australian range. Metamorph body size is highly plastic in this species, and our laboratory studies showed that larger metamorphs had better locomotor performance (both on land and in the water), and were more adept at catching and consuming prey. In mark-recapture trials in outdoor enclosures, larger body size enhanced metamorph survival and growth rate under some seasonal conditions. Larger metamorphs maintained their size advantage over smaller siblings for at least a month. Our data support the critical but rarely-tested assumption that all else being equal, larger body size at metamorphosis is likely to enhance an individual's long term viability. Thus, manipulations to reduce body size at metamorphosis in cane toads may help to reduce the ecological impact of this invasive species.

Exploiting intraspecific competitive mechanisms to control invasive cane toads (Rhinella marina).

If invasive species use chemical weapons to suppress the viability of conspecifics, we may be able to exploit those species-specific chemical cues for selective control of the invader. Cane toads (Rhinella marina) are spreading through tropical Australia, with negative effects on native species. The tadpoles of cane toads eliminate intraspecific competitors by locating and consuming newly laid eggs. Our laboratory trials show that tadpoles find those eggs by searching for the powerful bufadienolide toxins (especially, bufogenins) that toads use to deter predators. Using those toxins as bait, funnel-traps placed in natural waterbodies achieved near-complete eradication of cane toad tadpoles with minimal collateral damage (because most native (non-target) species are repelled by the toads' toxins). More generally, communication systems that have evolved for intraspecific conflict provide novel opportunities for invasive-species control.

Embryonic exposure to conspecific chemicals suppresses cane toad growth and survival.

Adaptations to suppress the viability of conspecifics may provide novel ways to control invasive taxa. The spread of cane toads (Rhinella marina) through tropical Australia has had severe ecological impacts, stimulating a search for biocontrol. Our experiments show that cane toad tadpoles produce waterborne chemical cues that suppress the viability of conspecifics encountering those cues during embryonic development. Brief (72 h) exposure to these cues in the egg and post-hatching phases massively reduced rates of survival and growth of larvae. Body sizes at metamorphosis (about three weeks later) were almost twice as great in control larvae as in tadpole-exposed larvae. The waterborne cue responsible for these effects might provide a weapon to reduce toad recruitment within the species' invaded range.

Ontogenetic variation in the chemical defenses of cane toads (Bufo marinus): toxin profiles and effects on predators.

We conducted a quantitative and qualitative chemical analysis of cane toad bufadienolides--the cardioactive steroids that are believed to be the principal cane toad toxins. We found complex shifts in toxin composition through toad ontogeny: (1) eggs contain at least 28 dominant bufadienolides, 17 of which are not detected in any other ontogenetic stage; (2) tadpoles present a simpler chemical profile with two to eight dominant bufadienolides; and (3) toxin diversity decreases during tadpole life but increases again after metamorphosis (larger metamorph/juvenile toads display five major bufadienolides). Total bufadienolide concentrations are highest in eggs (2.64 +/- 0.56 micromol/mg), decreasing during tadpole life stages (0.084 +/- 0.060 micromol/mg) before rising again after metamorphosis (2.35 +/- 0.45 micromol/mg). These variations in total bufadienolide levels correlate with toxicity to Australian frog species. For example, consumption of cane toad eggs killed tadpoles of two Australian frog species (Limnodynastes convexiusculus and Litoria rothii), whereas no tadpoles died after consuming late-stage cane toad tadpoles or small metamorphs. The high toxicity of toad eggs reflects components in the egg itself, not the surrounding jelly coat. Our results suggest a dramatic ontogenetic shift in the danger that toads pose to native predators, reflecting rapid changes in the types and amounts of toxins during toad development.

Impact of the invasive cane toad (Bufo marinus) on an Australian frog (Opisthodon ornatus) depends on minor variation in reproductive timing.

Invasive species are widely viewed as unmitigated ecological catastrophes, but the reality is more complex. Theoretically, invasive species could have negligible or even positive effects if they sufficiently reduce the intensity of processes regulating native populations. Understanding such mechanisms is crucial to predicting ultimate ecological impacts. We used a mesocosm experiment to quantify the impact of eggs and larvae of the introduced cane toad (Bufo marinus) on fitness-related traits (number, size and time of emergence of metamorphs) of a native Australian frog species (Opisthodon ornatus). The results depended upon the timing of oviposition of the two taxa, and hence the life-history stages that came into contact. Growth and survival of O. ornatus tadpoles were enhanced when they preceded B. marinus tadpoles into ponds, and reduced when they followed B. marinus tadpoles into ponds, relative to when tadpoles of both species were added to ponds simultaneously. The dominant tadpole-tadpole interaction is competition, and the results are consistent with competitive priority effects. However, these priority effects were reduced or reversed when O. ornatus tadpoles encountered B. marinus eggs. Predation on toxic toad eggs reduced the survival of O. ornatus and B. marinus. The consequent reduction in tadpole densities allowed the remaining O. ornatus tadpoles to grow more rapidly and to metamorphose at larger body sizes (>60% disparity in mean mass). Thus, exposure to B. marinus eggs reduced the number of O. ornatus metamorphs, but increased their body sizes. If the increased size at metamorphosis more than compensates for the reduced survival, the effective reproductive output of native anurans may be increased rather than decreased by the invasive toad. Minor interspecific differences in the seasonal timing of oviposition thus have the potential to massively alter the impact of invasive cane toads on native anurans.