Taste aversion training can educate free-ranging crocodiles against toxic invaders.

Apex predators play critical ecological roles, making their conservation a high priority. In tropical Australia, some populations of freshwater crocodiles (Crocodylus johnstoni) have plummeted by greater than 70% due to lethal ingestion of toxic invasive cane toads (Rhinella marina). Laboratory-based research has identified conditioned taste aversion (CTA) as a way to discourage consumption of toads. To translate those ideas into landscape-scale management, we deployed 2395 baits (toad carcasses with toxin removed and containing a nausea-inducing chemical) across four gorge systems in north-western Australia and monitored bait uptake with remote cameras. Crocodile abundance was quantified with surveys. Free-ranging crocodiles rapidly learned to avoid toad baits but continued to consume control (chicken) baits. Toad invasion at our sites was followed by high rates of crocodile mortality (especially for small individuals) at a control site but not at nearby treatment sites. In areas with high connectivity to other waterbodies, repeated baiting over successive years had continuing positive impacts on crocodile survival. In summary, we succeeded in buffering the often-catastrophic impact of invasive cane toads on apex predators.

A biological invasion modifies the dynamics of a host-parasite arms race.

By imposing novel selection pressures on both participants, biological invasions can modify evolutionary 'arms races' between hosts and parasites. A spatially replicated cross-infection experiment reveals strong spatial divergence in the ability of lungworms (Rhabdias pseudosphaerocephala) to infect invasive cane toads (Rhinella marina) in Australia. In areas colonized for longer than 20 years, toads are more resistant to infection by local strains of parasites than by allopatric strains. The situation reverses at the invasion front, where super-infective parasites have evolved. Invasion-induced shifts in genetic diversity and selective pressures may explain why hosts gain advantage over parasites in long-colonized areas, whereas parasites gain advantage at the invasion front.

Captivity induces large and population-dependent brain transcriptomic changes in wild-caught cane toads (Rhinella marina).

Gene expression levels are key molecular phenotypes at the interplay between genotype and environment. Mounting evidence suggests that short-term changes in environmental conditions, such as those encountered in captivity, can substantially affect gene expression levels. Yet, the exact magnitude of this effect, how general it is, and whether it results in parallel changes across populations are not well understood. Here, we take advantage of the well-studied cane toad, Rhinella marina, to examine the effect of short-term captivity on brain gene expression levels, and determine whether effects of captivity differ between long-colonized and vanguard populations of the cane toad's Australian invasion range. We compared the transcriptomes of wild-caught toads immediately assayed with those from toads captured from the same populations but maintained in captivity for seven months. We found large differences in gene expression levels between captive and wild-caught toads from the same population, with an over-representation of processes related to behaviour and the response to stress. Captivity had a much larger effect on both gene expression levels and gene expression variability in toads from vanguard populations compared to toads from long-colonized areas, potentially indicating an increased plasticity in toads at the leading edge of the invasion. Overall, our findings indicate that short-term captivity can induce large and population-specific transcriptomic changes, which has significant implications for studies comparing phenotypic traits of wild-caught organisms from different populations that have been held in captivity.

Brain transcriptome analysis reveals gene expression differences associated with dispersal behaviour between range-front and range-core populations of invasive cane toads in Australia.

Understanding the mechanisms allowing invasive species to adapt to novel environments is a challenge in invasion biology. Many invaders demonstrate rapid evolution of behavioural traits involved in range expansion such as locomotor activity, exploration and risk-taking. However, the molecular mechanisms that underpin these changes are poorly understood. In 86 years, invasive cane toads (Rhinella marina) in Australia have drastically expanded their geographic range westward from coastal Queensland to Western Australia. During their range expansion, toads have undergone extensive phenotypic changes, particularly in behaviours that enhance the toads' dispersal ability. Common-garden experiments have shown that some changes in behavioural traits related to dispersal are heritable. At the molecular level, it is currently unknown whether these changes in dispersal-related behaviour are underlain by small or large differences in gene expression, nor is known the biological function of genes showing differential expression. Here, we used RNA-seq to gain a better understanding of the molecular mechanisms underlying dispersal-related behavioural changes. We compared the brain transcriptomes of toads from the Hawai'ian source population, as well as three distinct populations from across the Australian invasive range. We found markedly different gene expression profiles between the source population and Australian toads. By contrast, toads from across the Australian invasive range had very similar transcriptomic profiles. Yet, key genes with functions putatively related to dispersal behaviour showed differential expression between populations located at each end of the invasive range. These genes could play an important role in the behavioural changes characteristic of range expansion in Australian cane toads.

In an arms race between host and parasite, a lungworm's ability to infect a toad is determined by host susceptibility not parasite preference.

Evolutionary arms races can alter both parasite infectivity and host resistance, and it is difficult to separate the effects of these twin determinants of infection outcomes. We used a co-introduced, invasive host-parasite system (the lungworm Rhabdias pseudosphaerocephala and cane toads Rhinella marina), where rapid adaptation and dispersal have led to population differences in infection resistance. We quantified behavioural responses of parasite larvae to skin-chemical cues of toads from different invasive populations, and rates at which juvenile hosts became infected following standardized exposure to lungworms. Chemical cues from toad skin altered host-seeking behaviour by parasites, similarly among populations. The number of infection attempts (parasite larvae entering the host's body) also did not differ between populations, but rates of successful infection (establishment of adult worm in host lungs) were higher for range-edge toads than for range-core conspecifics. Thus, lower resistance to parasite infection in range-edge juvenile toads appears to be due to less effective immune defences of the host rather than differential behavioural responses of the parasite. In this ongoing host-parasite arms race, changing outcomes appear to be driven by shifts in host immunocompetence.

Wildfires modify the parasite loads of invasive cane toads.

The frequency and severity of wildfires are increasing due to anthropogenic modifications to habitats and to climate. Post-fire landscapes may advantage invasive species via multiple mechanisms, including changes to host-parasite interactions. We surveyed the incidence of endoparasitic lungworms (Rhabdias pseudosphaerocephala) in invasive cane toads (Rhinella marina) in near-coastal sites of eastern Australia, a year after extensive fires in this region. Both the prevalence of infection and number of worms in infected toads increased with toad body size in unburned areas. By contrast, parasite load decreased with toad body size in burned areas. By killing moisture-dependent free-living lungworm larvae, the intense fires may have liberated adult cane toads from a parasite that can substantially reduce the viability of its host. Smaller toads, which are restricted to moist environments, did not receive this benefit from fires.

The accelerating anuran: evolution of locomotor performance in cane toads (Rhinella marina, Bufonidae) at an invasion front.

As is common in biological invasions, the rate at which cane toads (Rhinella marina) have spread across tropical Australia has accelerated through time. Individuals at the invasion front travel further than range-core conspecifics and exhibit distinctive morphologies that may facilitate rapid dispersal. However, the links between these morphological changes and locomotor performance have not been clearly documented. We used raceway trials and high-speed videography to document locomotor traits (e.g. hop distances, heights, velocities, and angles of take-off and landing) of toads from range-core and invasion-front populations. Locomotor performance varied geographically, and this variation in performance was linked to morphological features that have evolved during the toads' Australian invasion. Geographical variation in morphology and locomotor ability was evident not only in wild-caught animals, but also in individuals that had been raised under standardized conditions in captivity. Our data thus support the hypothesis that the cane toad's invasion across Australia has generated rapid evolutionary shifts in dispersal-relevant performance traits, and that these differences in performance are linked to concurrent shifts in morphological traits.

Urbanization and translocation disrupt the relationship between host density and parasite abundance.

The species interactions that structure natural communities are increasingly disrupted by radical habitat change resulting from the widespread processes of urbanization and species translocations. Although many species are disadvantaged by these changes, others thrive in these new environments, achieving densities that exceed those in natural habitats. Often the same species that benefit from urbanization are successful invaders in introduced habitats, suggesting that similar processes promote these species in both environments. Both processes may especially benefit certain species by modifying their interactions with harmful parasites ('enemy release'). To detect such modifications, we first need to identify the mechanisms underlying host-parasite associations in natural populations, then test whether they are disrupted in cities and introduced habitats. We studied the interaction between the cane toad Rhinella marina, a globally invasive species native to South America, and its Amblyomma ticks. Our field study of 642 cane toads across 46 sites within their native range in French Guiana revealed that 56% of toads carried ticks, and that toads with ticks were in poor body condition relative to uninfected conspecifics. Across natural and disturbed habitats tick prevalence and abundance increased with toad density, but this association was disrupted in the urban environment, where tick abundance remained low even where toad densities were high, and prevalence decreased with density. Reductions in the abundance of ticks in urban habitats may be attributable to pesticides (which are sprayed for mosquito control but are also lethal to ticks), and our literature review shows that tick abundance is generally lower in cane toads from urban habitats across South America. In the invasive range, ticks were either absent (in 1,960 toads from Puerto Rico, Hawai'i, Japan and Australia) or less abundant (in Florida and the Caribbean; literature review). The positive relationship between host density and parasite abundance is thought to be a key mechanism through which parasites regulate host populations; anthropogenic processes that disrupt this relationship may allow populations in urban and introduced habitats to persist at densities that would otherwise lead to severe impacts from parasites.

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.

May the (selective) force be with you: Spatial sorting and natural selection exert opposing forces on limb length in an invasive amphibian.

Spatial sorting on invasion fronts drives the evolution of dispersive phenotypes, and in doing so can push phenotypes in the opposite direction to natural selection. The invasion of cane toads (Rhinella marina) through tropical Australia has accelerated over recent decades because of the accumulation of dispersal-enhancing traits at the invasion front, driven by spatial sorting. One such trait is the length of the forelimbs: invasion-front toads have longer arms (relative to body length) in comparison with populations 10-20 years after invasion. Such a shift likely has fitness consequences: an increase of forearm length would decrease the strength with which a male could cling to a female during amplexus and so render such a male less competitive in competition for mates, compared to short-armed conspecifics. Our laboratory trials of attachment strength confirmed that males with relatively longer arms were easier to displace, and competition trials show higher duration of amplexus for males with shorter arms. Together with the sharp cline in limb length observed behind the invasion front, these results imply an opposition of selective forces: spatial sorting optimizes dispersal, but as this force wanes behind the invasion front, we see the primacy of natural selection reassert itself.

At the invasion front, male cane toads (Rhinella marina) have smaller testes.

As a colonizing species expands its range, individuals at the invasion front experience different evolutionary pressures than do those at the range-core. For example, low densities at the edge of the range mean that males should rarely experience intense sperm competition from rivals; and investment into reproduction may trade-off with adaptations for more rapid dispersal. Both of these processes are predicted to favour a reduction in testis size at the invasion front. To explore effects of invasion stage in Australian cane toads (Rhinella marina), we collected and dissected 214 adult males from three regions: one in the species' range-core (northeastern Australia), and two from invasion fronts (one in northwestern Australia and one in southeastern Australia). Despite the brief duration of separation between toads in these areas (approx. 85 years), testis masses averaged greater than 30% higher (as a proportion of body mass) in range-core males than in conspecifics sampled from either vanguard of the invasion. Previous work has documented low reproductive frequencies in female cane toads at the invasion front also, consistent with the hypothesis that evolutionary and ecological pressures unleashed by an invasion can favour relatively low resource allocation to reproduction in both sexes.

Non-reproductive male cane toads (Rhinella marina) withhold sex-identifying information from their rivals.

A male cane toad (Rhinella marina) that mistakenly clasps another male (rather than a female) in a sexual embrace (amplexus) can be induced to dismount by a male-specific 'release call'. Although that sex-identifying system can benefit both males in that interaction, our standardized tests showed that one-third of male cane toads did not emit release calls when grasped. Most of those silent males were small, had small testes relative to body mass, and had poorly developed secondary sexual characteristics. If emitting a release call is costly (e.g. by attracting predators), a non-reproductive male may benefit by remaining silent; other cues (such as skin rugosity) will soon induce the amplexing male to dismount, and the 'opportunity cost' to being amplexed (inability to search for and clasp a female) is minimal for non-reproductive males. Hence, male toads may inform other males about their sexual identity only when it is beneficial to do so.

Cane toads (Rhinella marina) rely on water access, not drought tolerance, to invade xeric Australian environments.

The invasion of habitats with novel environmental challenges may require physiological tolerances not seen in conspecifics from the native range. We used a combination of field and laboratory-based experiments to assess physiological tolerance to limited water access at four sites distributed across the historical invasion path of cane toads (Rhinella marina) in Australia that, from east to west, alternated between mesic and seasonally xeric habitats. Toads from all locations were well hydrated at the time of capture. However, experimental dehydration caused greater mass loss, higher plasma osmolality, and inhibition of lytic ability in toads from xeric compared to mesic locations. These results suggest somewhat surprisingly that toads from xeric environments are physiologically more vulnerable to water loss. In contrast, bactericidal ability was not sensitive to hydric state and was greater in toads from eastern (long-colonized) areas. Similar patterns in lytic ability in hydrated toads and agglutination ability in wild toads suggest that toads along the invasion front face a tradeoff between enhanced dispersal ability and physiological responses to dehydration. The ability of this invasive species to spread into drier environments may be underpinned by a combination of phenotypic plasticity and evolved (heritable) traits.

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.

Pathology Associated With an Outbreak of Entamoebiasis in Wild Cane Toads (Rhinella marina) in Tropical Australia.

Infection due to Entamoeba spp. is known to cause serious disease in primates (Entamoeba histolytica) and snakes (Entamoeba invadens), but there are no detailed descriptions of the pathology associated with Entamoeba spp. infection in amphibians. In 2014, an outbreak of entamoebiasis associated with a novel species of Entamoeba induced clinical illness and poor body condition in free-ranging cane toads in Australia's Northern Territory. Here, we describe the gross pathology, histology, and clinical pathology linked to the outbreak. The study compared 25 toads with invasive entamoebiasis, defined as histologically visible amoebas within tissue, and 12 toads without invasive entamoebiasis. Grossly, affected toads had mild to marked congestion of colonic serosal vasculature, with variable thickening of the intestinal wall and serosanguineous to hemorrhagic colonic content. Histologically, invasive entamoebiasis manifested primarily as moderate to severe, variably hyperplastic to ulcerative colitis. The small intestine was affected in 10 of 25 toads, and 5 of 25 toads also had gastric lesions. Amoebas consistent in morphology with Entamoeba sp. were commonly intermingled with mucosal epithelium, frequently along the basement membrane, with deeper invasion into the superficial lamina propria in only 5 toads. Toads with invasive entamoebiasis had neutrophilia, monocytosis, and lymphopenia, and thus elevated neutrophil to lymphocyte ratios, suggestive of an inflammatory and/or stress leukogram.

Invasive Colonic Entamoebiasis in Wild Cane Toads, Australia.

We detected a disease syndrome in free-ranging Australian cane toads involving atypical behavior and emaciation that is associated with a previously undescribed Entamoeba sp. that infiltrates the colonic lining, causing it to slough. The organism may become seasonally pathogenic when toads are under hydric and nutritional stress.

Anuran predators overcome visual illusion: dazzle coloration does not protect moving prey.

Predators everywhere impose strong selection pressures on the morphology and behavior of their prey, but the resulting antipredator adaptations vary greatly among species. Studies of adaptive coloration in prey species have generally focused on cryptic or aposematic prey, with little consideration of color patterns in palatable mobile prey. Complex color patterns have been proposed to decrease the ability of visual predators to capture moving prey (motion dazzle effect). Most support for this hypothesis, however, comes from experiments with human subjects and simulated prey. We tested the motion dazzle effect using, for the first time, natural predators (cane toads, Rhinella marina) and live prey (house crickets, Acheta domesticus) with altered color patterns. We found no support for the motion dazzle effect as striped crickets did not fare better than solid colored ones. Crickets that spent more time moving, however, were more likely to be eaten. Our results suggest that motion specialized visual predators such as toads overcome the motion dazzle effect and impose stronger selection pressure on prey behavior than on coloration. These findings emphasize the importance of sensory specializations of predators in mediating antipredator strategies.

Exploratory behavior of a native anuran species with high invasive potential.

Exploratory behavior can be a key component of survival in novel or changing environments, ultimately determining population establishment. While many studies have investigated the behavior of wild animals in response to novel food items or objects, our understanding of how they explore novel environments is limited. Here, we examine how experience affects the foraging behavior of a species with high invasive potential. In particular, we investigate the movement and behavior of cane toads as a function of experience in a novel environment, and how the presence of food modulates exploration. Cane toads, from a population in their native range, were repeatedly tested in a large, naturalistic arena with or without food present. Both groups exhibited significant but different changes in exploratory behavior. While toads in an environment without food reduced exploratory behavior over trials, those with food present increased both food intake per trial and the directness of their paths to food, resulting in fewer approaches to food patches over time. Our results suggest that cane toads learn patch location and provide preliminary evidence suggesting toads use spatial memory, not associative learning, to locate food. In sum, we show that with experience, cane toads alter their behavior to increase foraging efficiency. This study emphasizes the role of learning in foraging in cane toads, a characteristic that may have facilitated their success as invaders.

Invasion history alters the behavioural consequences of immune system activation in cane toads.

Acute activation of the immune system often initiates a suite of behavioural changes. These "sickness behaviours"-involving lethargy and decreased activity-may be particularly costly on invasion fronts, where evolutionary pressures on dispersal favour individuals that move large distances. We used a combination of field and laboratory studies to compare sickness behaviours of cane toads from populations differing in invasion history. To do this we stimulated immune system activation by injecting lipopolysaccharide (LPS) to mimic bacterial infection. We predicted that LPS would result in less severe sickness behaviour in toads from range-edge populations because they had undergone selection for rapid and sustained dispersal (activities in conflict with lethargy and decreased activity). Contrary to our prediction, LPS injection caused a greater reduction in dispersal-relevant traits in invasion-front individuals than in conspecifics from the range-core. Our data suggest that the rapid invasion of cane toads through tropical Australia has seen an evolutionary shift in the magnitude of sickness behaviour elicited by pathogen infection. The increased sickness behaviour among range-edge toads suggests a shift away from pathogen tolerance (seen in range-core populations) towards resistance to pathogen attack. But as a consequence, when pathogens do become successfully established, toads from invasion-front populations may have less capacity to tolerate their ill-effects.

The loneliness of the long-distance toad: invasion history and social attraction in cane toads (Rhinella marina).

Individuals at the leading edge of a biological invasion constantly encounter novel environments. These pioneers may benefit from increased social attraction, because low population densities reduce competition and risks of pathogen transfer, and increase benefits of information transfer. In standardized trials, cane toads (Rhinella marina) from invasion-front populations approached conspecifics more often, and spent more time close to them, than did conspecifics from high-density, long-colonized populations.