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?'

Colonization history affects heating rates of invasive cane toads.

Amphibians in hot climates may be able to avoid high temperatures by controlling their rates of heating. In northern Australia, invasive cane toads (Rhinella marina) experience hot dry conditions in newly-colonized (western) sites but milder conditions in longer-occupied (eastern) sites. Under standardized conditions, toads from western sites heated less rapidly than did conspecifics from an eastern site. The availability of free water slowed heating rates of eastern but not western toads. Thus, the colonization of climatically extreme sites has been accompanied by a rapid shift in the toads' ability to remain cool under hot conditions, even when free water is not available.

Exposure of cane toad hatchlings to older conspecifics suppresses chemosensory food tracking behaviour and increases risk of predation post-exposure.

Attempts to control invasive species using species-specific pheromones need to incorporate an understanding of interactive effects among those pathways. The larvae of invasive cane toads (Rhinella marina) utilise chemical cues to repulse, attract or suppress conspecific larvae. We can exploit these effects to reduce toad abundance, but the effects of each cue may not be additive. That is, exposure to one type of cue may lessen the impact of exposure to another cue. To assess this possibility, we exposed toad larvae to combinations of cues. Tadpoles that had been exposed to the suppression cue during larval development exhibited no response to the attraction cue, resulting in lower capture rates in attractant-baited traps. Suppression, however, did not affect a tadpole's response to the alarm cue, and exposure to the alarm cue during tadpole development did not affect response to the attraction cue. Tadpoles exposed to the suppression cue were smaller than control tadpoles at 10 days post-exposure, and consequently were more vulnerable to gape-limited invertebrate predators. Our results demonstrate that the responses by toad tadpoles to chemical cues interact in important ways, and are not simply additive when combined. Control efforts need to incorporate an understanding of such interactions if we are to most effectively use chemical-communication pathways to control invasive amphibians.

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.

The cost of chemical defence: the impact of toxin depletion on growth and behaviour of cane toads ( Rhinella marina).

Many animals capable of deploying chemical defences are reluctant to use them, suggesting that synthesis of toxins imposes a substantial cost. Typically, such costs have been quantified by measuring the elevation in metabolic rate induced by toxin depletion (i.e. during replenishment of toxin stores). More generally, we might expect that toxin depletion will induce shifts in a broad suite of fitness-relevant traits. In cane toads ( Rhinella marina), toxic compounds that protect against predators and pathogens are stored in large parotoid (shoulder) glands. We used correlational and experimental approaches in field and laboratory settings to investigate impacts of toxin depletion on growth rate and behaviour in cane toads. In free-ranging toads, larger toxin stores were associated with smaller gonads and livers, suggesting energetic trade-offs between toxin production and both reproduction and energy metabolism. Experimental removal of toxin (by manually squeezing parotoid glands) reduced rates of growth in body mass in both captive and free-ranging toads. Radio tracking demonstrated that de-toxined toads dispersed more slowly than did control toads. Given that toxin stores in cane toads take several months to fully replenish, deploying toxin to repel a predator may impose a substantial cost, explaining why toads use toxin only as a final line of defence.

The cane or marine toad, Rhinella marina (Anura, Bufonidae): two genetically and morphologically distinct species.

Rhinella marina is a Neotropical toad that has been introduced widely worldwide. Its toxic effects to frog-eating predators threaten the native and domestic fauna of some regions where it has been introduced. Despite previous studies suggesting two genetically distinct cryptic species within R. marina, one east and one west of the Andes, its taxonomic status remained unresolved due to the absence of morphological complementary evidence. For the first time, data from two mitochondrial genes (ND3 and CR) and 23 morphometric landmarks are combined to evaluate the taxonomic status of this species. Our results support the hypothesis of two separate evolutionary lineages within R. marina and demonstrate that these lineages have significantly diverged in skull shape. We identified two distinct morphotypes, one eastern and one Andean western, with no overlapping morphospaces. The geographic pattern of genetic variation was consistent with a stable structured population with no evidence of recent demographic or geographic expansions. The concordance between the observed geographic patterns in morphometric and genic traits calls for the recognition of two species under R. marina name.

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.

Living up to its name? The effect of salinity on development, growth, and phenotype of the "marine" toad (Rhinella marina).

The highly permeable integument of amphibians renders them vulnerable to chemical characteristics of their environment, especially during the aquatic larval stage. As the cane toad (Rhinella marina, Bufonidae) invades southwards along the east coast of Australia, it is encountering waterbodies with highly variable conditions of temperature, pH, and salinity. Understanding the tolerance of toads to these conditions can clarify the likely further spread of the invader, as well as the adaptability of the species to novel environmental challenges. We measured salinity in waterbodies in the field and conducted laboratory trials to investigate the impacts of salinity on toad viability. Eggs and tadpoles from the southern invasion front tolerated the most saline conditions we found in potential spawning ponds during surveys [equivalent to 1200 ppm (3.5 % the salinity of seawater)]. Indeed, high-salinity treatments increased tadpole body sizes, accelerated metamorphosis, and improved locomotor ability of metamorphs (but did not affect metamorph morphology). At very low salinity [40 ppm (0.1 % seawater)], eggs hatched but larvae did not develop past Gosner stage 37. Our study shows that the egg and larval life stages of cane toads can tolerate wide variation in the salinity of natal ponds and that this aspect of waterbody chemistry is likely to facilitate rather than constrain continued southward expansion of the toad invasion front in eastern Australia.

Differences in developmental strategies between long-settled and invasion-front populations of the cane toad in Australia.

Phenotypic plasticity can enhance a species' ability to persist in a new and stressful environment, so that reaction norms are expected to evolve as organisms encounter novel environments. Biological invasions provide a robust system to investigate such changes. We measured the rates of early growth and development in tadpoles of invasive cane toads (Rhinella marina) in Australia, from a range of locations and at different larval densities. Populations in long-colonized areas have had the opportunity to adapt to local conditions, whereas at the expanding range edge, the invader is likely to encounter challenges that are both novel and unpredictable. We thus expected invasion-vanguard populations to exhibit less phenotypic plasticity than range-core populations. Compared to clutches from long-colonized areas, clutches from the invasion front were indeed less plastic (i.e. rates of larval growth and development were less sensitive to density). In contrast, those rates were highly variable in clutches from the invasion front, even among siblings from the same clutch under standard conditions. Clutches with highly variable rates of growth and development under constant conditions had lower phenotypic plasticity, suggesting a trade-off between these two strategies. Although these results reveal a strong pattern, further investigation is needed to determine whether these different developmental strategies are adaptive (i.e. adaptive phenotypic plasticity vs. bet-hedging) or instead are driven by geographic variation in genetic quality or parental effects.

The Acid Test: pH Tolerance of the Eggs and Larvae of the Invasive Cane Toad (Rhinella marina) in Southeastern Australia.

Invasive cane toads are colonizing southeastern Australia via a narrow coastal strip sandwiched between unsuitable areas (Pacific Ocean to the east, mountains to the west). Many of the available spawning sites exhibit abiotic conditions (e.g., temperature, salinity, and pH) more extreme than those encountered elsewhere in the toad's native or already invaded range. Will that challenge impede toad expansion? To answer that question, we measured pH in 35 ponds in northeastern New South Wales and 8 ponds in the Sydney region, in both areas where toads occur (and breed) and adjacent areas where toads are likely to invade, and conducted laboratory experiments to quantify effects of pH on the survival and development of toad eggs and larvae. Our field surveys revealed wide variation in pH (3.9-9.8) among natural water bodies. In the laboratory, the hatching success of eggs was increased at low pH (down to pH 4), whereas the survival, growth, and developmental rates of tadpoles were enhanced by higher pH levels. We found that pH influenced metamorph size and shape (relative head width, relative leg length) but not locomotor performance. The broad tolerance range of these early life-history stages suggests that pH conditions in ponds will not significantly slow the toad's expansion southward. Indeed, toads may benefit from transiently low pH conditions, and habitat where pH in wetlands is consistently low (such as coastal heath) may enhance rather than reduce toad reproductive success. A broad physiological tolerance during embryonic and larval life has contributed significantly to the cane toad's success as a widespread colonizer.

The Effects of a Nematode Lungworm (Rhabdias hylae) on its Natural and Invasive Anuran Hosts.

Biological invasions can bring both the invader and native taxa into contact with novel parasites. As cane toads ( Rhinella marina ) have spread through Australia, they have encountered lungworms (Rhabdias hylae) that occur in native frogs. Field surveys suggest that these lungworms have not host-switched to toads. In our laboratory studies, R. hylae infected cane toads as readily as it infected native frogs, but failed to reach the lungs of the novel host (i.e., were killed by the toads' immune response). Plausibly, then, R. hylae might reduce the viability both of their native hosts (frogs, that can exhibit high parasite burdens) and cane toads (that must deal with infective larvae traveling through the host body). Our laboratory trials suggest, however, that the impacts of the parasite on infected anuran hosts (both frogs and toads) were minimal, with no significant decrements to host survival, activity, growth, or locomotor performance. Ironically, the lack of impact of the parasite on its native hosts appears to be an outcome of co-evolution (frogs tolerate the lungworm), whereas the lack of impact on the novel host is due to a lack of co-evolution (toads can recognize and eliminate the lungworm).

The effects of the toxic cyanobacterium Limnothrix (strain AC0243) on Bufo marinus larvae.

Limnothrix (strain AC0243) is a cyanobacterium, which has only recently been identified as toxin producing. Under laboratory conditions, Bufo marinus larvae were exposed to 100,000 cells mL(-1) of Limnothrix (strain AC0243) live cultures for seven days. Histological examinations were conducted post mortem and revealed damage to the notochord, eyes, brain, liver, kidney, pancreas, gastrointestinal tract, and heart. The histopathological results highlight the toxicological impact of this strain, particularly during developmental stages. Toxicological similarities to ß-N-Methylamino-L-alanine are discussed.

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.

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.

The effects of experimentally infecting Australian tree frogs with lungworms (Rhabdias pseudosphaerocephala) from invasive cane toads.

Invasive species may transmit novel pathogens to native taxa, and lacking a history of coevolutionary interactions with the pathogen, the new hosts may be severely affected. Cane toads (Rhinella marina) were introduced to Australia in 1935, bringing with them a lungworm (Rhabdias pseudosphaerocephala) not found in Australian frogs. Previous studies suggest that most frog species are unaffected by this parasite, but one tree-frog (Litoria caerulea) can harbour high numbers of lungworm. More detailed laboratory studies confirm and extend the earlier results on L. caerulea and show that Rhabdias infection severely depresses the viability of metamorphs of an allied tree-frog species, Litoria splendida. Parasitic larvae infected both of these two closely related tree-frog species, but the two anurans differed in the consequences of infection. Parasitism reduced the survivorship of L. splendida and the stamina of both species. Lungworms did not consistently reduce growth rates or affect heart rates in either tree-frog species. Although L. splendida is potentially vulnerable to the arrival of toad-transported lungworms, rates of host-switching may be reduced by low levels of habitat overlap between the frogs (which are rock-dwelling and arboreal) and the toads (which are terrestrial and most abundant in disturbed habitats).

The evolution of growth rates on an expanding range edge.

Individuals in the vanguard of a species invasion face altered selective conditions when compared with conspecifics behind the invasion front. Assortment by dispersal ability on the expanding front, for example, drives the evolution of increased dispersal, which, in turn, leads to accelerated rates of invasion. Here I propose an additional evolutionary mechanism to explain accelerating invasions: shifts in population growth rate (r). Because individuals in the vanguard face lower population density than those in established populations, they should (relative to individuals in established populations) experience greater r-selection. To test this possibility, I used the ongoing invasion of cane toads (Bufo marinus) across northern Australia. Life-history theory shows that the most efficient way to increase the rate of population growth is to reproduce earlier. Thus, I predict that toads on the invasion front will exhibit faster individual growth rates (and thus will reach breeding size earlier) than those from older populations. Using a common garden design, I show that this is indeed the case: both tadpoles and juvenile toads from frontal populations grow around 30 per cent faster than those from older, long established populations. These results support theoretical predictions that r increases during range advance and highlight the importance of understanding the evolution of life history during range advance.

Rhabdias pseudosphaerocephala infection in Bufo marinus: lung nematodes reduce viability of metamorph cane toads.

Cane toads (Bufo marinus) were introduced to Australia in 1935 and have since spread widely over the continent, generating concern regarding ecological impacts on native predators. Most Australian cane toad populations are infected with lung nematodes Rhabdias pseudosphaerocephala, a parasite endemic to New World (native-range) cane toad populations; presumably introduced to Australia with its toad host. Considering the high intensities and prevalence reached by this parasite in Australian toad populations, and public ardour for developing a control plan for the invasive host species, the lack of experimental studies on this host-parasite system is surprising. To investigate the extent to which this lungworm influences cane toad viability, we experimentally infected metamorph toads (the smallest and presumably most vulnerable terrestrial phase of the anuran life cycle) with the helminth. Infected toads exhibited reduced survival and growth rates, impaired locomotor performance (both speed and endurance), and reduced prey intake. In summary, R. pseudosphaerocephala can substantially reduce the viability of metamorph cane toads.

Factors influencing responses to alarm pheromone by larvae of invasive cane toads, Bufo marinus.

If pheromonal communication systems of invasive species differ from those of native biota, it may be possible to control the invader by exploiting that difference. When injured, the larvae of cane toads, Bufo marinus, an invasive species of major concern in tropical Australia, produce species-specific chemical cues that alert conspecific tadpoles to danger. Repeated exposure to the alarm chemical reduces tadpole survival rates and body sizes at metamorphosis and, thus, could help control toad populations. To evaluate the feasibility of this approach, we need to know how the intensity of toad tadpole response to the alarm chemical is affected by factors such as water temperature, time of day, larval stage and feeding history, geographic origin of the tadpoles, and habituation. Information on these topics may enable us to optimize deployment, so that tadpoles encounter pheromone at the times and places that confer maximum effect. In our studies, tadpole density, nutritional state, larval stage, and geographic origin had little effect on the intensity of the alarm response, but tadpoles reacted most strongly in higher water temperatures and during daylight hours. Repeated, once-daily exposure to pheromone did not induce habituation, but repeated exposure at 15-min intervals did not elicit further responses after 2 h total exposure. The insensitivity of response to most factors tested means that the effectiveness of the pheromone as a control agent should be relatively robust.

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.

Cane toad toxicity: an assessment of extracts from early developmental stages and adult tissues using MDCK cell culture.

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g., eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 microM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.