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 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.

The adaptive significance of large size at birth in marine snakes.

Evolutionary shifts from one habitat type to another can clarify selective forces that affect life-history attributes. Four lineages of snakes (acrochordids and three clades within the Elapidae) have invaded marine habitats, and all have larger offspring than do terrestrial snakes. Predation by fishes on small neonates offers a plausible selective mechanism for that shift, because ascending to breathe at the ocean surface exposes a marine snake to midwater predation whereas juvenile snakes in terrestrial habitats can remain hidden. Consistent with this hypothesis, snake-shaped models moving through a coral-reef habitat in New Caledonia attracted high rates of attack by predatory fishes, and small models (the size of neonatal terrestrial snakes) were attacked more frequently than were large models (the size of neonatal sea snakes). Vulnerability to predatory fishes may have imposed strong selection for increased offspring size in marine snakes.

Foraging mode constrains the evolution of cephalic horns in lizards and snakes.

A phylogenetically diverse minority of snake and lizard species exhibit rostral and ocular appendages that substantially modify the shape of their heads. These cephalic horns have evolved multiple times in diverse squamate lineages, enabling comparative tests of hypotheses on the benefits and costs of these distinctive traits. Here, we demonstrate correlated evolution between the occurrence of horns and foraging mode. We argue that although horns may be beneficial for various functions (e.g. camouflage, defence) in animals that move infrequently, they make active foragers more conspicuous to prey and predators, and hence are maladaptive. We therefore expected horns to be more common in species that ambush prey (entailing low movement rates) rather than in actively searching (frequently moving) species. Consistent with that hypothesis, our phylogenetic comparative analysis of published data on 1939 species reveals that cephalic horns occur almost exclusively in sit-and-wait predators. This finding underlines how foraging mode constrains the morphology of squamates and provides a compelling starting point for similar studies in other animal groups.

Can nesting behaviour allow reptiles to adapt to climate change?

A range of abiotic parameters within a reptile nest influence the viability and attributes (including sex, behaviour and body size) of hatchlings that emerge from that nest. As a result of that sensitivity, a reproducing female can manipulate the phenotypic attributes of her offspring by laying her eggs at times and in places that provide specific conditions. Nesting reptiles shift their behaviour in terms of timing of oviposition, nest location and depth of eggs beneath the soil surface across spatial and temporal gradients. Those maternal manipulations affect mean values and variances of both temperature and soil moisture, and may modify the vulnerability of embryos to threats such as predation and parasitism. By altering thermal and hydric conditions in reptile nests, climate change has the potential to dramatically modify the developmental trajectories and survival rates of embryos, and the phenotypes of hatchlings. Reproducing females buffer such effects by modifying the timing, location and structure of nests in ways that enhance offspring viability. Nonetheless, our understanding of nesting behaviours in response to climate change remains limited in reptiles. Priority topics for future studies include documenting climate-induced changes in the nest environment, the degree to which maternal behavioural shifts can mitigate climate-related deleterious impacts on offspring development, and ecological and evolutionary consequences of maternal nesting responses to climate change. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Rapidly evolved traits enable new conservation tools: perspectives from the cane toad invasion of Australia.

Natural populations can show rapid adaptive responses to intense (human-mediated) environmental change. The potential for exploiting rapidly evolved traits for conservation management has been often discussed but rarely implemented. Capitalizing on a well-studied biological invasion, we here explore the idea that rapid phenotypic change in the invaders, their pathogens, and the native biota provide opportunities for managers to control invader abundance and buffer adverse impacts on native wildlife. Intensive studies of the invasion of tropical Australia by cane toads (Rhinella marina) have identified newly evolved vulnerabilities that we could exploit for toad control; and newly evolved resilience of native wildlife that we could exploit for impact reduction. For example, distinctive phenotypes of toads at the expanding range edge enhance dispersal rate but reduce reproductive output, intraspecific competitive ability, and immunocompetence; and the evolution of larval cannibalism creates opportunities not only for species-specific trapping of toad tadpoles, but also could be exploited (when allied to emerging CRISPR-Cas9 techniques) to intensify intraspecific conflict in invasive toads. That is, we could use the invasive species to control their own populations. This case study illustrates the potential of detailed basic research to identify novel approaches for conservation.

Do changes in body mass alter white blood cell profiles and immune function in Australian cane toads (Rhinella marina)?

Variation in food resources can result in dramatic fluctuations in the body condition of animals dependent on those resources. Decreases in body mass can disrupt patterns of energy allocation and impose stress, thereby altering immune function. In this study, we investigated links between changes in body mass of captive cane toads (Rhinella marina), their circulating white blood cell populations, and their performance in immune assays. Captive toads that lost weight over a three-month period had increased levels of monocytes and heterophils and reduced levels of eosinophils. Basophil and lymphocyte levels were unrelated to changes in mass. Because individuals that lost mass had higher heterophil levels but stable lymphocyte levels, the ratio of these cell types was also higher, partially consistent with a stress response. Phagocytic ability of whole blood was higher in toads that lost mass, owing to increased circulating levels of phagocytic cells. Other measures of immune performance were unrelated to mass change. These results highlight the challenges faced by invasive species as they expand their range into novel environments which may impose substantial seasonal changes in food availability that were not present in the native range. Individuals facing energy restrictions may shift their immune function towards more economical and general avenues of combating pathogens. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

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.

Carrying eggs uphill: are costs of reproduction stronger on steeper slopes?

Locomotor impairment during pregnancy is a well-documented cost of reproduction, but most empirical studies have not incorporated ecological complexity, such as locomotion on sloping inclines rather than horizontal surfaces. Biomechanical factors suggest that carrying a heavy burden-including shifts in the body's centre of mass-may impair locomotor ability even more when an animal is running uphill. If so, then measuring costs of reproduction on horizontal racetracks may underestimate these costs in nature for arboreal species. To evaluate this prediction, we measured the pregnancy-induced reduction in speed for jacky dragons (Amphibolurus muricatus) at inclines ranging from 0 to 45°. Both pregnancy and steeper slopes reduced lizard performance, but pregnancy did not exacerbate the locomotor decrement on steeper racetracks. An ability to maintain mobility on steep slopes during pregnancy may be a target of selection in arboreal taxa. To understand the evolutionary context of locomotion-based costs of reproduction, we also need studies on the relationship between organismal performance and ecologically relevant measures such as predation risk.

Geographic variation in bacterial assemblages on cane toad skin is influenced more by local environments than by evolved changes in host traits.

Bacterial assemblages on amphibian skin may play an important role in protecting hosts against infection. In hosts that occur over a range of environments, geographic variation in composition of bacterial assemblages might be due to direct effects of local factors and/or to evolved characteristics of the host. Invasive cane toads (Rhinella marina) are an ideal candidate to evaluate environmental and genetic mechanisms, because toads have evolved major shifts in physiology, morphology, and behavior during their brief history in Australia. We used samples from free-ranging toads to quantify site-level differences in bacterial assemblages and a common-garden experiment to see if those differences disappeared when toads were raised under standardised conditions at one site. The large differences in bacterial communities on toads from different regions were not seen in offspring raised in a common environment. Relaxing bacterial clustering to operational taxonomic units in place of amplicon sequence variants likewise revealed high similarity among bacterial assemblages on toads in the common-garden study, and with free-ranging toads captured nearby. Thus, the marked geographic divergence in bacterial assemblages on wild-caught cane toads across their Australian invasion appears to result primarily from local environmental effects rather than evolved shifts in the host.

Sexual dimorphism and reproductive biology of the Asian bockadam snake (Cerberus schneiderii) in West Java.

Although they are among the most abundant snakes on Earth, and are heavily exploited for their skins and meat, Asian bockadams (or "dog-faced water snakes", Cerberus schneiderii) have attracted relatively little study across their wide geographic range. Based on dissection of 3,382 snakes brought to processing facilities in and around the city of Cirebon in West Java, Indonesia, we document facets of the biology of these mangrove-dwelling aquatic homalopsids. Females attain larger body sizes than do males, and are heavier-bodied (due in part to greater fat reserves) but have shorter tails relative to snout-vent length. Males showed testicular enlargement late in the year (August-November) but both reproductive and non-reproductive females were found year-round. Litters were large (3 to 45 offspring), especially in larger females. The commercial harvest falls mainly on adult snakes of both sexes, with seasonal variation in sex ratios. Life-history traits such as early maturation and frequent production of large litters render this species resilient to commercial harvesting. Future research should explore reasons for strong variation among facilities in the sex ratios of snakes, potentially identifying ways to focus the harvest on the sex (males) whose numbers are less critical for population viability.

The banded colour patterns of sea snakes discourage attack by predatory fishes, enabling Batesian mimicry by harmless species.

The evolution of bright 'warning' colours in nontoxic animals often is attributed to mimicry of toxic species, but empirical tests of that hypothesis must overcome the logistical challenge of quantifying differential rates of predation in nature. Populations of a harmless sea snake species (Emydocephalus annulatus) in New Caledonia exhibit colour polymorphism, with around 20% of individuals banded rather than melanic. Stability in that proportion over 20 years has been attributed to Batesian mimicry of deadly snake species by banded morphs of the harmless taxon. This hypothesis requires that banded colours reduce a snake's vulnerability to predation. We tested that idea by pulling flexible snake-shaped models through the water and recording responses by predatory fish. Black and banded lures attracted similar numbers of following fish, but attacks were directed almost exclusively to black lures. Our methods overcome several ambiguities associated with experimental studies on mimicry in terrestrial snakes and support the hypothesis that banded colour patterns reduce a non-venomous marine snake's vulnerability to predation.

Taking the bait: Developing a bait delivery system to target free-ranging crocodiles and varanid lizards with a novel conservation strategy.

In tropical Australia, conditioned taste aversion (CTA) can buffer vulnerable native predators from the invasion of a toxic prey species (cane toads, Rhinella marina). Thus, we need to develop methods to deploy aversion-inducing baits in the field, in ways that maximize uptake by vulnerable species (but not other taxa). We constructed and field-tested baiting devices, in situ with wild animals. Apparatus were set next to waterbodies and baited concurrently at multiple locations (over water, water's edge, and on the bank). Baits were checked and replaced twice daily during the trial; remote cameras recorded visitation by native predators. Bait longevity was compared at sun-exposed and shaded locations over 12 h. The strength required to remove baits from apparatus was measured in varanids and crocodiles. The device promoted high rates of bait uptake by freshwater crocodiles (47% baits consumed), varanid lizards (19% baits consumed), and non-target taxa (34% baits consumed). Targeting specific predators can be achieved by manipulating bait location and time of deployment, as well as the force required to dislodge the bait. Crocodiles were best targeted with over-water baits, whereas varanid lizards preferred baits located at the edges of waterbodies. When testing bait longevity in ambient conditions, during the daytime baits desiccated fully within 12 h, and faster in the sun than in the shade. Based on studies using captive animals, the "pulling force" strength of reptilian predators scaled with body size and was greater in crocodiles than in varanid lizards. We present the first conservation baiting protocol designed specifically for reptiles. Our results demonstrate the feasibility of widespread and taxon-specific deployment of aversion-inducing baits to buffer the impacts of invasive cane toads, and our methods are applicable (with modification) to other research and management programs globally.

Sex-based differences in the use of post-fire habitats by invasive cane toads (Rhinella marina).

Wildfires can modify habitat attributes, and those changes may differentially affect males versus females within a species if there is pre-existing niche divergence between the sexes. We used radio-tracking and dissections to study invasive cane toads (Rhinella marina), and performed transect counts on native frogs and cane toads 12 months after extensive fires in forests of eastern Australia. Both toads and native frogs were encountered more frequently in burned sites than in unburned sites. Most microhabitat features were similar between burned versus unburned areas, but fire had differential impacts on the ecology of male versus female toads. In burned areas females were less numerous but were larger, in better body condition, and had consumed more prey (especially, coleopterans and myriapods). The impact of fire on attributes of retreat-sites (e.g., temperature, density of vegetation cover) also differed between the sexes. More generally, intraspecific divergence in ecological traits within a species (as a function of body size as well as sex) may translate into substantial divergences in the impacts of habitat change.

No evidence for cold-adapted life-history traits in cool-climate populations of invasive cane toads (Rhinella marina).

As an invasive organism spreads into a novel environment, it may encounter strong selective pressures to adapt to abiotic and biotic challenges. We examined the effect of water temperature during larval life on rates of survival and growth of the early life-history stages of cane toads (Rhinella marina) from two geographic regions (tropical vs. temperate) in the species' invaded range in eastern Australia. If local adaptation at the southern (cool-climate) invasion front has extended the cold-tolerance of early life-stages, we would expect to see higher viability of southern-population toads under cooler conditions. Our comparisons revealed no such divergence: the effects of water temperature on rates of larval survival and growth, time to metamorphosis, size at metamorphosis and locomotor performance of metamorphs were similar in both sets of populations. In two cases where tropical and temperate-zone populations diverged in responses to temperature, the tropical animals performed better at low to medium temperatures than did conspecifics from cooler regions. Adaptation to low temperatures in the south might be constrained by behavioural shifts (e.g., in reproductive seasonality, spawning-site selection) that allow toads to breed in warmer water even in cool climates, by gene flow from warmer-climate populations, or by phylogenetic conservatism in these traits.

Spatial ecology, activity patterns, and habitat use by giant pythons (Simalia amethistina) in tropical Australia.

Although giant snakes are abundant in some tropical forests, their ecology is far less well-known than for smaller species of snakes in cooler climates. Information on spatial ecology can clarify management issues such as the sizes and types of habitats needed for conservation. We radio-tracked 27 scrub pythons (Simalia amethistina; snout-vent lengths 2.02 to 3.70 m) in Cape York, near the northeastern tip of Australia, for a mean period of 426 days (up to 1001 days) per snake. Home ranges were larger in males than females (means 0.60 vs. 0.28 km2) and overlapped considerably among individuals. All snakes used rainforest habitat, but seasonal shifts into open woodland were common. Snakes were active primarily by night, with larger snakes hunting less of the time overall, and more often by day. Hunting behaviour was seen more often during the wet season than the dry season. Average daily displacement was < 10 m, typically involving a shift from diurnal refuge to nocturnal ambush-site. A reliance on sit-and-wait predation results in small home ranges and limited movements, despite the large body size of this species.

Divergence in life-history traits among three adjoining populations of the sea snake Emydocephalus annulatus (Hydrophiinae, Elapidae).

Life-history traits such as rates of growth, survival and reproduction can vary though time within a single population, or through space among populations, due to abiotically-driven changes in resource availability. In terrestrial reptiles, parameters such as temperature and rainfall generate variation in life-histories-but other parameters likely are more important in marine systems. We studied three populations of sea snakes (Emydocephalus annulatus) in adjacent bays in the IndoPacific archipelago of New Caledonia. The extreme philopatry of individual snakes allows us to unambiguously allocate each animal to one of the three populations. Although water temperatures and rainfall do not differ over this small scale, one site experiences more intense winds, restricting opportunities for foraging. Our 18-year mark-recapture dataset (> 1,200 snakes, > 2,400 captures) reveals significant divergence among populations in life-history traits. Survival rates and population densities were similar among sites, but snakes at the most wind-exposed site (Anse Vata) exhibited lower body condition, slower growth, less frequent production of litters, and smaller litters. Weather-driven variation in feeding rates thus may affect life-history traits of marine snakes as well as their terrestrial counterparts, but driven by different parameters (e.g., wind exposure rather than variation in temperatures or rainfall).

Frequency-dependent Batesian mimicry maintains colour polymorphism in a sea snake population.

Evolutionary theory suggests that polymorphic traits can be maintained within a single population only under specific conditions, such as negative frequency-dependent selection or heterozygote advantage. Non-venomous turtle-headed sea snakes (Emydocephalus annulatus) living in shallow bays near Noumea in New Caledonia exhibit three colour morphs: black, black-and-white banded, and an intermediate (grey-banded) morph that darkens with age. We recorded morph frequencies during 18 consecutive years of surveys, and found that the numbers of recruits (neonates plus immigrants) belonging to each morph increased in years when that morph was unusually rare in the population, and decreased when that morph was unusually common. Thus, morph frequencies are maintained by negative frequency-dependent selection. We interpret the situation as Batesian mimicry of highly venomous sea snakes (Aipysurus, Hydrophis, Laticauda) that occur in the same bays, and range in colour from black-and-white banded to grey-banded. Consistent with the idea that mimicry may protect snakes from attack by large fish and sea eagles, behavioural studies have shown that smaller fish species in these bays flee from banded snakes but attack black individuals. As predicted by theory, mimetic (banded) morphs are less common than the cryptically-coloured melanic morph.