Isolation breeds naivety : island living robs Australian varanid lizards of toad-toxin immunity via four-base-pair mutation

Since their introduction to the toad-free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian–African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1-H2 domain of the α1 subunit of the sodium-potassium-ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian–African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1-H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian–African H1-H2 domains. The transfections resulted in an approximate 3000-fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian–African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

Do the numbers and locations of road-killed anuran carcasses accurately reflect impacts of vehicular traffic?

Road-killed animals are easy and inexpensive to survey, and may provide information about species distributions, abundances, and mortality rates. As with any sampling method, however, we need to explore methodological biases in such data. First, how does an animal's behavior (e.g., use of the center vs. periphery of the road) influence its vulnerability to vehicular traffic? Second, how rapidly do post-mortem processes (scavenging by other animals, destruction or displacement by subsequent vehicles) change the numbers and locations of roadkills? Our surveys of anurans on a highway in tropical Australia show that different anuran species are distributed in different ways across the width of the road, and that locations of live versus dead animals sometimes differ within a species. Experimental trials show that location on the road affects the probability of being hit by a vehicle, with anurans in the middle of the road begin hit 35% more often than anurans on the edges; thus, center-using species are more likely to be hit than edge-using taxa. The magnitude of post-mortem displacement and destruction by subsequent vehicles depended on anuran species and body size. The mean parallel displacement distance was 122.7 cm, and carcasses of thin-skinned species exhibited greater post-mortem destruction. Scavenging raptors removed 73% of carcasses, most within a few hours of sunrise. Removal rates were biased with respect to size and species. Overall, our studies suggest that investigators should carefully evaluate potential biases before using roadkill counts to estimate underlying animal abundances or mortality rates.

Artificial water points facilitate the spread of an invasive vertebrate in arid Australia

Summary: The spread of invasive species after their initial introduction is often facilitated by human actions. In some cases, invaders only become established in habitats where dominant native species have been displaced as a result of human actions or where humans inadvertently provide essential resources such as food, water or shelter. We investigated if dams that provide water for livestock have facilitated the cane toad's (Rhinella marina) invasion of a hot semi-arid landscape by providing toads with a resource subsidy and hence refuge from extreme heat and aridity. To determine the relationship between the presence of surface water and habitat occupancy by toads, we surveyed natural and artificial water features for cane toads during the annual dry season. We used radiotracking and acoustic tags to determine whether movement patterns and shelter use of cane toads were focussed around dams. To determine whether dams provide toads with refuge from extreme heat and aridity, we deployed plaster models with internal thermometers to estimate ambient temperatures and toad desiccation rates in shelter sites. To determine whether dams alleviate the stress experienced by toads, we measured plasma corticosterone levels of toads that sheltered in and away from dams. Toads were present in sites with standing water and absent from waterless sites. Most radiotracked toads sheltered within 1 m of water. Toad movements were focussed around water. Toads tracked with passive acoustic telemetry over a 6-month dry season were highly resident at dams. Plaster models placed in toad shelter sites away from the water lost 27% more mass and experienced higher temperatures than models placed near the water's edge. Toads that sheltered in terrestrial shelters exhibited higher plasma corticosterone levels compared to toads that sheltered near dams. Dams provide toads with refuge habitats where they are less at risk from overheating and dehydration. Synthesis and applications. Artificial water points can facilitate biological invasions in arid regions by providing a resource subsidy for water-dependent invasive species. Our study suggests that there is scope to control populations of water-dependent invasive vertebrates in arid regions by restricting their access to artificial water points.

Behavioural flexibility allows an invasive vertebrate to survive in a semi-arid environment

Plasticity or evolution in behavioural responses are key attributes of successful animal invasions. In northern Australia, the invasive cane toad (Rhinella marina) recently invaded semi-arid regions. Here, cane toads endure repeated daily bouts of severe desiccation and thermal stress during the long dry season (April-October). We investigated whether cane toads have shifted their ancestral nocturnal rehydration behaviour to one that exploits water resources during the day. Such a shift in hydration behaviour could increase the fitness of individual toads by reducing exposure to desiccation and thermal stress suffered during the day even within terrestrial shelters. We used a novel method (acoustic tags) to monitor the daily hydration behaviour of 20 toads at two artificial reservoirs on Camfield station, Northern Territory. Remarkably, cane toads visited reservoirs to rehydrate during daylight hours, with peaks in activity between 9.00 and 17.00. This diurnal pattern of rehydration activity contrasts with nocturnal rehydration behaviour exhibited by adult toads in their native geographical range and more mesic parts of Australia. Our results demonstrate that cane toads phase shift a key behaviour to survive in a harsh semi-arid landscape. Behavioural phase shifts have rarely been reported in invasive species but could facilitate ongoing invasion success.

Interplay among nocturnal activity, melatonin, corticosterone and performance in the invasive cane toad (Rhinella marinus)

Most animals conduct daily activities exclusively either during the day or at night. Here, hormones such as melatonin and corticosterone, greatly influence the synchronization or regulation of physiological and behavioral cycles needed for daily activity. How then do species that exhibit more flexible daily activity patterns, responses to ecological, environmental or life-history processes, regulate daily hormone profiles important to daily performance? This study examined the consequences of (1) nocturnal activity on diel profiles of melatonin and corticosterone and (2) the effects of experimentally increased acute melatonin levels on physiological and metabolic performance in the cane toad (Rhinella marinus). Unlike inactive captive toads that had a distinct nocturnal melatonin profile, nocturnally active toads sampled under field and captive conditions, exhibited decreased nocturnal melatonin profiles with no evidence for any phase shift. Nocturnal corticosterone levels were significantly higher in field active toads than captive toads. In toads with experimentally increased melatonin levels, plasma lactate and glucose responses following recovery post exercise were significantly different from control toads. However, exogenously increased melatonin did not affect resting metabolism in toads. These results suggest that toads could adjust daily hormone profiles to match nocturnal activity requirements, thereby avoiding performance costs induced by high nocturnal melatonin levels. The ability of toads to exhibit plasticity in daily hormone cycles, could have broad implications for how they and other animals utilize behavioral flexibility to optimize daily activities in response to natural and increasingly human mediated environmental variation.

Adrenocortical stress responses influence an invasive vertebrate's fitness in an extreme environment

Continued range expansion into physiologically challenging environments requires invasive species to maintain adaptive phenotypic performance. The adrenocortical stress response, governed in part by glucocorticoid hormones, influences physiological and behavioural responses of vertebrates to environmental stressors. However, any adaptive role of this response in invasive populations that are expanding into extreme environments is currently unclear. We experimentally manipulated the adrenocortical stress response of invasive cane toads (Rhinella marina) to investigate its effect on phenotypic performance and fitness at the species' range front in the Tanami Desert, Australia. Here, toads are vulnerable to overheating and dehydration during the annual hot-dry season and display elevated plasma corticosterone levels indicative of severe environmental stress. By comparing unmanipulated control toads with toads whose adrenocortical stress response was manipulated to increase acute physiological stress responsiveness, we found that control toads had significantly reduced daily evaporative water loss and higher survival relative to the experimental animals. The adrenocortical stress response hence appears essential in facilitating complex phenotypic performance and setting fitness trajectories of individuals from invasive species during range expansion.

Landscape context influences chytrid fungus distribution in an endangered European amphibian

Wildlife disease is an emerging threat to biodiversity. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been documented in over 500 amphibian species globally. Understanding conditions under which amphibians are vulnerable to Bd is important for evaluating species risk and developing surveillance strategies. Here, we investigate the spatial distribution of Bd infection in the ephemeral pond-breeding yellow-bellied toad Bombina variegata, a species of high conservation concern in the European Union. We sampled 550 toads from 60 ponds in a traditional agricultural landscape in Southern Transylvania, Romania. Overall, Bd prevalence was low in B.variegata, but infected toads were widely dispersed through the landscape and were found in a quarter of all sampled ephemeral ponds. At the pond level, increased Bd occurrence was associated with short distances to perennial water sources and high forest cover. These findings suggest that perennial water sources may act as source habitat for Bd, with amphibian movements resulting in Bd spillover into ephemeral ponds. Increased Bd occurrence in ponds surrounded by high levels of forest cover is likely related to cooler and wetter conditions that are more favourable for Bd. Throughout the study landscape, patchy environmental suitability for Bd appears to restrict the pathogen to a subset of B.variegata habitat. Ephemeral ponds in open landscapes, without nearby perennial habitat, likely provide an environmental refuge from Bd, where the risk of infection is low. From a conservation perspective, these findings highlight the importance of maintaining ephemeral ponds in open landscapes, but these are currently threatened by land-use change.

Invasive cane toad triggers chronic physiological stress and decreased reproductive success in an island endemic

Understanding the mechanisms that afford invasive species their ecological success as important agents of global change is key to addressing their biodiversity impacts. Species invasions that occur on small islands are especially detrimental and suggest that invaders intensify their ecological impacts by exploiting novel ecological functions. However, it remains unknown whether such strong impacts are also a consequence of an invader's indirect effect (e.g. causing physiological stress or reproductive failure) on island species. Therefore, it is valuable to quantify the physiological mechanisms through which invasive species can exert indirect effects on the performance, and ultimately the fitness of island endemics. In this study, we investigated whether the invasive cane toad (Rhinella marina) caused indirect competitive impacts on the endemic Fijian ground frog (Platymantis vitiana) on the small (60 ha) Viwa Island, Fiji. We used large (4 × 10 000 m2), natural and replicated enclosures to monitor ground frog stress hormone levels, reproductive hormone cycle, body condition, breeding and survival in the presence/absence of the cane toad. We conducted monthly sampling to analyse annual patterns in testosterone for males, estradiol and progesterone for females, corticosterone for both sexes and body condition of ground frogs in replicated enclosures or natural habitats with high/low cane toad densities. We also measured survival and reproductive success of ground frogs in enclosures. Results showed that in both enclosures and natural habitats with high cane toad densities, ground frogs had a significant reduction in body condition, increased urinary corticosterone metabolites and suppressed sex steroid metabolites. Most importantly, annual field surveys showed significant reduction in ground frog reproductive success (fewer eggs were laid in enclosures with toads present); however, survival was not severely reduced. Our study clearly demonstrated that on small islands, invasive species may exploit broader ecological roles with strong indirect effects that amplify their impacts beyond those seen on continents. Overall, the effects of cane toad competition had the capacity to strongly reduce ground frog reproductive success. We strongly advocate management actions that either minimize invasion or limit the strength of invasive-native species interactions (e.g. through habitat conservation) to prevent further extinctions on islands.

Restricting access to invasion hubs enables sustained control of an invasive vertebrate

Biological invasions often occur through expansion of satellite populations that become established at 'invasion hubs'. Invasion hubs can result from random dispersal events, but frequently arise when invading individuals actively choose habitats using cues that signify high-quality environments where the fitness consequences are positive. Theoretical studies suggest that targeted control at invasion hubs can effectively suppress the populations and impacts of invaders. In arid Australia, small dams that provide water for livestock function as invasion hubs by providing an invasive vertebrate, the cane toad Rhinella marina, with refuge from extreme aridity during the annual dry season. Toads are attracted to dams and use them as stepping stone habitats from which they disperse during rainy periods. Here, we ask whether sustained control of this invasive vertebrate can be achieved by converting invasion hubs into ecological traps. We did this by manipulating invasion hub habitats to induce a mismatch between toads' habitat preference and the fitness consequences of their habitat choice to cause high mortality. We constructed fences to exclude toads from dams and maintained these fences for 1 year. This period encompassed periods of dry and wet seasonal climatic conditions. Our manipulation did not alter the attractive cues for invading toads which died en masse while attempting to settle at fenced dams that prevented toads from reaching water. Toad populations at the fenced dams were suppressed by 1-2 orders of magnitude compared to unfenced controls and procedural controls. Toad populations remained suppressed for a year after exclusion. By excluding toads from dams, we converted invasion hubs into ecological traps and effectively thwarted the reinvasion of cane toads. Our research suggests that water exclusion devices could be used to prevent toad invasion or to control cane toad populations in arid landscapes colonized by toads. Synthesis and applications. Our study demonstrates that sustained control of invader populations can be achieved by restricting their access to invasion hubs. Control of invasive species via elimination of invasion hubs could be conducted reactively, to control established populations of invaders, or conducted strategically, by rendering invasion hubs unsuitable for colonization ahead of the invasion front to prevent further population spread.

Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na(+)/K(+)-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na(+)/K(+)-ATPase H1-H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na(+)/K(+)-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.