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.

Whole-mitogenome analysis unveils previously undescribed genetic diversity in cane toads across their invasion trajectory.

Invasive species offer insights into rapid adaptation to novel environments. The iconic cane toad (Rhinella marina) is an excellent model for studying rapid adaptation during invasion. Previous research using the mitochondrial NADH dehydrogenase 3 (ND3) gene in Hawai'ian and Australian invasive populations found a single haplotype, indicating an extreme genetic bottleneck following introduction. Nuclear genetic diversity also exhibited reductions across the genome in these two populations. Here, we investigated the mitochondrial genomics of cane toads across this invasion trajectory. We created the first reference mitochondrial genome for this species using long-read sequence data. We combined whole-genome resequencing data of 15 toads with published transcriptomic data of 125 individuals to construct nearly complete mitochondrial genomes from the native (French Guiana) and introduced (Hawai'i and Australia) ranges for population genomic analyses. In agreement with previous investigations of these populations, we identified genetic bottlenecks in both Hawai'ian and Australian introduced populations, alongside evidence of population expansion in the invasive ranges. Although mitochondrial genetic diversity in introduced populations was reduced, our results revealed that it had been underestimated: we identified 45 mitochondrial haplotypes in Hawai'ian and Australian samples, none of which were found in the native range. Additionally, we identified two distinct groups of haplotypes from the native range, separated by a minimum of 110 base pairs (0.6%). These findings enhance our understanding of how invasion has shaped the genetic landscape of this species.

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.

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

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

Increased rates of dispersal of free-ranging cane toads (Rhinella marina) during their global invasion.

Invasions often accelerate through time, as dispersal-enhancing traits accumulate at the expanding range edge. How does the dispersal behaviour of individual organisms shift to increase rates of population spread? We collate data from 44 radio-tracking studies (in total, of 650 animals) of cane toads (Rhinella marina) to quantify distances moved per day, and the frequency of displacement in their native range (French Guiana) and two invaded areas (Hawai'i and Australia). We show that toads in their native-range, Hawai'i and eastern Australia are relatively sedentary, while toads dispersing across tropical Australia increased their daily distances travelled from 20 to 200 m per day. That increase reflects an increasing propensity to change diurnal retreat sites every day, as well as to move further during each nocturnal displacement. Daily changes in retreat site evolved earlier than did changes in distances moved per night, indicating a breakdown in philopatry before other movement behaviours were optimised to maximise dispersal.

Discovery of Novel Viruses Associated With the Invasive Cane Toad (Rhinella marina) in Its Native and Introduced Ranges.

Cane toads (Rhinella marina) are notoriously successful invaders: from 101 individuals brought to Australia in 1935, poisonous toads now cover an area >1.2 million km2 with adverse effects on native fauna. Despite extensive research on the role of macroparasites in cane toad invasion, viral research is lagging. We compared viral prevalence and diversity between toads in their native range (French Guiana, n=25) and two introduced ranges: Australia (n=151) and Hawai'i (n=10) with a metatranscriptomic and metagenomic approach combined with PCR screening. Australian toads almost exclusively harbor one of seven viruses detected globally. Rhimavirus-A (Picornaviridae) exhibited low genetic diversity and likely actively infected 9% of sampled Australian toads extending across ~2,000km of Northern Australia and up to the current invasion front. In native range cane toads, we identified multiple phylogenetically distinct viruses (Iridoviridae, Picornaviridae, Papillomaviridae, and Nackedna-like virus). None of the same viruses was detected in both ranges, suggesting that Australian cane toads have largely escaped the viral infection experienced by their native range counterparts. The novel native range viruses described here are potential biocontrol agents, as Australian toads likely lack prior immunological exposure to these viruses. Overall, our evidence suggests that there may be differences between viruses infecting cane toads in their native vs. introduced ranges, which lays the groundwork for further studies on how these viruses have influenced the toads' invasion history.

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

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

Spatial ecology of cane toads (Rhinella marina) in their native range: a radiotelemetric study from French Guiana.

Like most invasive species, cane toads have attracted less research in their native range than in invaded areas. We radio-tracked 34 free-ranging toads in French Guiana, a source region for most invasive populations, across two coastal and two rainforest sites. Coastal toads generally sheltered in pools of fresh or brackish water but nocturnally foraged on beaches, whereas rainforest toads sheltered in forested habitats, moving into open areas at night. Over five days of monitoring, native toads frequently re-used shelters and moved little between days (means = 10-63 m/site) compared to invasion-front toads from Australia (~ 250 m). Larger toads moved less between days, but displaced in more consistent directions. At night, foraging toads travelled up to 200 m before returning to shelters. Foraging distance was related to body condition at coastal sites, with toads in poorer body condition travelling farther. Rain increased the probability of coastal toads sheltering in the dry habitats where they foraged. Dispersal and rainfall were lower at coastal sites, and the strategies utilized by coastal toads to minimize water loss resembled those of invasive toads in semi-desert habitats. This global invader already exhibits a broad environmental niche and substantial behavioural flexibility within its native range.

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?'

Diurnal activity in cane toads (Rhinella marina) is geographically widespread.

Although adult cane toads (Rhinella marina) are generally active only at night, a recent study reported that individuals of this species switched to diurnal activity in response to encountering a novel habitat type (deeply shaded gorges) in the course of their Australian invasion. Our sampling over a broader geographic scale challenges the idea that this behaviour is novel; we documented diurnal behaviour both in the species' native range and in several sites within the invaded range, in multiple habitat types. Diurnal activity was most common in the tropics and in areas where toads attain high population densities and are in poor body condition, suggesting that the expansion of activity times may be induced by intraspecific competition for food.

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.

Increased Adaptive Variation Despite Reduced Overall Genetic Diversity in a Rapidly Adapting Invader.

Invasive species often evolve rapidly following introduction despite genetic bottlenecks that may result from small numbers of founders; however, some invasions may not fit this "genetic paradox". The invasive cane toad (Rhinella marina) displays high phenotypic variation across its introduced Australian range. Here, we used three genome-wide datasets to characterize their population structure and genetic diversity. We found that toads form three genetic clusters: 1) native range toads, 2) toads from the source population in Hawaii and long-established areas near introduction sites in Australia, and 3) toads from more recently established northern Australian sites. Although we find an overall reduction in genetic diversity following introduction, we do not see this reduction in loci putatively under selection, suggesting that genetic diversity may have been maintained at ecologically relevant traits, or that mutation rates were high enough to maintain adaptive potential. Nonetheless, toads encounter novel environmental challenges in Australia, and the transition between genetic clusters occurs at a point along the invasion transect where temperature rises and rainfall decreases. We identify environmentally associated loci known to be involved in resistance to heat and dehydration. This study highlights that natural selection occurs rapidly and plays a vital role in shaping the structure of invasive populations.

Sex and weaponry: The distribution of toxin-storage glands on the bodies of male and female cane toads (Rhinella marina).

The parotoid macroglands of bufonid anurans store (and can expel) large volumes of toxic secretions and have attracted detailed research. However, toxins also are stored in smaller glands that are distributed on the limbs and dorsal surface of the body. Female and male cane toads (Rhinella marina) differ in the location of toxin-storage glands and the extent of glandular structures. Female toads store a larger proportion of their toxins in the parotoids than males as well as (to a lesser extent) in smaller glands on the forelimbs. Males have smaller and more elongate parotoids than females, but glands cover more of the skin surface on their limbs (especially hindlimbs) and dorsal surface. The delay to toxin exudation in response to electrostimulation varied among glands in various parts of the body, and did so differently in males than in females. The spatial distribution of toxin glands differs between the sexes even in toads that have been raised under standardized conditions in captivity; hence, the sexual dimorphism is due to heritable factors rather than developmentally plastic responses to ecological (e.g., habitat, predation risk) differences between the sexes. The selective advantages of this sexual dimorphism remain unclear. A priori, we might expect to see toxin widely dispersed across any part of the body likely to be contacted by a predator; and a wide distribution also would be expected if the gland secretions have other (e.g., male-male rivalry) functions. Why, then, is toxin concentrated in the parotoids, especially in female toads? That concentration may enhance the effectiveness of frontal displays to deter predation and also may facilitate the transfer of stored toxins to eggs.

Grass invasion increases top-down pressure on an amphibian via structurally mediated effects on an intraguild predator.

Plants serve as both basal resources and ecosystem engineers, so plant invasion may exert trophic influences on consumers both via bottom-up processes and by altering the environmental context in which trophic interactions occur. To determine how these mechanisms affect a native predator we used a mark-recapture study in eight pairs of 58-m2 field enclosures to measure the influence of Japanese stilt grass invasion on 3200 recently metamorphosed American toads. Toad survivorship was lower in invaded habitats despite abiotic effects that favor amphibians. Prey densities were also lower in invaded habitats, but growth was unaffected. Frequent spider predation events in invaded habitats led us to use factorial field cage manipulations of stilt grass and lycosid spiders to determine if invasion increases predation rates. Spiders persisted at higher densities in the presence of stilt grass, and toad survival was lowest in cages with both grass and spiders. Invasion alone did not significantly reduce toad survival. Our results demonstrate that despite prey reductions and abiotic effects, it is increased spider persistence that reduces toad survival in invaded habitats. Invasion therefore affects resident forest floor consumers by modifying trophic interactions between native species, causing structurally mediated reductions in intraguild predation rates among spiders, with cascading implications for toad survival.

New findings from an old pathogen: intraerythrocytic bacteria (family Anaplasmatacea) in red-backed salamanders Plethodon cinereus.

During a recent study of red-backed salamanders (Plethodon cinereus), we discovered an intraerythrocytic organism typified by violet-staining, intracellular inclusions, consistent with descriptions of Cytamoeba or Aegyptianella (bacteria). Here we characterize its taxonomic status using molecular techniques and ask basic questions about its nature. Blood smears from 102 salamanders were examined from Pennsylvania, New York, and Virginia to determine prevalence, and whole blood from several infected animals was tested using a PCR which targets the 16S rRNA gene of bacteria. Phylogenetic analysis of partial 16S rRNA gene sequence (1201 bp) indicated this organism was in the order Rickettsiales and is likely a member of the family Anaplasmatacea. The organism differed from currently described taxa and was clearly differentiated from Aegyptianella pullorum of birds and "Candidatus Hemobacterium ranarum" (formally A. ranarum) of frogs. Of all salamanders, 17 (16.7%) were infected and these were significantly larger (snout-vent length) and had higher body condition scores than uninfected ones, and males were more likely to be infected than females. Erythrocytes affected by the pathogen were 5% larger than unaffected ones, but otherwise similar in morphology. Infected animals tended to have a greater number of circulating white blood cells, based on estimates from smears, indicating a nonspecific response to the pathogen by the innate immune system. Given its phylogenetic position, this pathogen is likely transmitted by an arthropod vector, and the male-biased prevalence strongly implicates trombiculid mites, which also live in leaf litter and affect male salamanders more so than females.