Invasive Toads Research Articles

Conditioned taste aversion enhances the survival of an endangered predator imperilled by a toxic invader

Summary 1. Even when we cannot eradicate an invasive species, we may be able to reduce its ecological impact. In Australia, a critically endangered predator, the northern quoll Dasyurus hallucatus is threatened by the invasion of the highly toxic cane toad Bufo marinus. Following toad invasion, quoll populations have become extinct across Northern Australia. Toads are continuing to spread, and will soon invade the Kimberley, one of the quoll’s last strongholds. To prevent future local extinctions, we need a new approach for mitigating the impact of cane toads on this iconic predator. 2. We investigated whether conditioned taste aversion (CTA) could be used to modify quoll predatory behaviour and mitigate toad impacts. We successfully induced an aversion to live toads in juvenile northern quolls by feeding them a dead toad containing a nausea-inducing chemical (thiabendazole). 3. To investigate whether CTA enhanced quoll survival, we fitted radiocollars to 31 toad-smart and

Factors affecting the vulnerability of cane toads (Bufo marinus) to predation by ants

Experimental evidence on the determinants of prey vulnerability is scarce, especially for vertebrates in the field. Invasive species offer robust opportunities to explore prey vulnerability, because the intensity of predation on or by such animals has not been eroded by coevolution. Around waterbodies in tropical Australia, native meat ants (Iridomyrmex reburrus) consume many metamorph cane toads (Bufo marinus, an invasive anuran). We document the determinants of toad vulnerability, especially the roles of toad body size and ant density. Larger metamorphs were attacked sooner (because they attracted more ants), but escaped more often. Overall, smaller toads were more likely to be killed. Ant densities influenced toad responses, as well as attack rate and success. Data on the immediate outcomes of attacks underestimate mortality: more than 73% of apparent ‘escapees’ died within 24 h. Because mortality during this period was independent of toad size, predation was less size selective than suggested by immediate outcomes. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 738–751.

Indirect ecological impacts of an invasive toad on predator–prey interactions among native species

One of the many ways that invasive species can affect native ecosystems is by modifying the behavioural and ecological interactions among native species. For example, the arrival of the highly toxic cane toad (Bufo marinus) in tropical Australia has induced toad-aversion in some native predators. Has that shift also affected the predators’ responses to native prey—for example, by reducing vulnerability of native tadpoles via a mimicry effect, or increasing vulnerability of other prey types (such as insects) via a shift in predator feeding tactics? We exposed a native predator (northern trout gudgeon, Mogurnda mogurnda) to toad tadpoles in the laboratory, and measured effects of that exposure on the fish’s subsequent intake of native tadpoles and crickets. As predicted, toad-exposed fishes reduced their rate of predation on (palatable) tadpoles of native frogs (Litoria caerulea and L. nasuta). If alternative prey (crickets) were available also, the toad-exposed fishes shifted even more strongly away from predation on native tadpoles. Thus, invasion of a toxic species can provide a mimicry benefit to native taxa that resemble the invader, and can shift predation pressure onto other taxa.

Using a native predator (the meat ant,Iridomyrmex reburrus) to reduce the abundance of an invasive species (the cane toad,Bufo marinus) in tropical Australia

Summary 1. Invasive species pose ecological threats in many areas, but attempts to control invaders by introducing other exotic species may cause further unanticipated problems. If we can use predators native to the introduced range to assist in control of the invader, the risks of collateral damage are lower. 2. In tropical Australia, high desiccation rates restrict newly-transformed (metamorph) cane toads Bufo marinus to the margins of waterbodies, rendering the metamorphs vulnerable to predatory ants (Iridomyrmex reburrus). By adding bait (catfood) to selected areas, we increased ant densities (and thus, toad mortality) more than fourfold. 3. Over 50% of attacks by ants in the field were immediately fatal to the metamorph toads, and most ‘escapee’ toads (88%) died of their injuries within 24 h after the attack. 4. When we increased ant densities by artificial baiting, 98% of metamorph toads were encountered, and 84% attacked, within the two-minute observation period. Collateral damage to native fauna appears to be low, but warrants closer examination. 5.Synthesis and applications. Manipulating the foraging locations of native predatory ants can substantially increase their off-take of invasive toads. More generally, vulnerabilities of invasive species to predators native to the introduced range may facilitate control of invader numbers with little collateral damage to the rest of the fauna.

Adjusting to a toxic invader: native Australian frogs learn not to prey on cane toads

Biological invasions provide opportunities to study novel behavioral interactions between predators and their prey. To withstand detrimental effects from a potentially lethal invader, a native taxon must somehow adjust to the invader’s presence. Cane toads (Bufo marinus) are highly toxic to native Australian anurans and constitute a major threat if consumed. We recorded the responses of Australian marbled frogs (Limnodynastes convexiusculus) during their first encounters with edible-sized cane toads. The frogs exhibited rapid avoidance learning: toad-exposed frogs were less likely to attack subsequently encountered cane toads (and hence more likely to survive). Among-clutch variance in learning rates and in physiological tolerance to toad toxins was low, suggesting that genetically based adaptive changes to frog feeding responses will be slow (especially given that rapid learning reduces mortality and thus reduces the fitness decrement of initial willingness to attack a toad). Hence, rapid taste aversion learning is the primary mechanism enabling marbled frogs to persist in the presence of a potentially fatal invader. In combination with previous work, our study shows that some native predators adjust to the threat posed by cane toad invasion via taste aversion learning, whereas others show genetically based modification of feeding responses. More generally, both learning and adaptation enable vulnerable native taxa to survive the arrival of a toxic invasive species. Key words: behavior, Bufo marinus, frog, invasive species, learning, predator. [Behav Ecol]

Impact of Invasive Cane Toads on Australian Birds

The cane toad (Bufo marinus), a large, toxic, American anuran, was introduced to Australia in 1935. Populations of many of Australia's reptiles (snakes, varanid lizards, crocodiles) and carnivorous mammals (dasyurid marsupials) have declined because these predators are killed by the toad's powerful toxins. In contrast to these well-studied species, little is known about the cane toads impacts on Australian birds. We reviewed published and unpublished data on behavioral interactions between Australian avian predators and cane toads and collated distributional and dietary information to identify avian taxa potentially at risk from cane toad invasion. Cane toads are sympatric with 172 frog-eating bird species in Australia, and an additional 8 bird species overlap with the predicted future range of the toad. Although many bird species thus are potentially at risk, behavioral observations suggest the risk level is generally low. Despite occasional reports of Australian birds being killed when they ingest cane toads, most birds either ignore toads or survive the predation event. The apparently higher tolerance of Australian birds to toad toxins, compared with Australian reptiles and marsupials, may reflect genetic exchange between Australian birds and Asian populations that encounter other bufonid species regularly and hence have evolved the capacity to recognize or tolerate this toxic prey.

Locomotor performance in an invasive species: cane toads from the invasion front have greater endurance, but not speed, compared to conspecifics from a long-colonised area

Cane toads (Bufo marinus) are now moving about 5 times faster through tropical Australia than they did a half-century ago, during the early phases of toad invasion. Radio-tracking has revealed higher daily rates of displacement by toads at the invasion front compared to those from long-colonised areas: toads from frontal populations follow straighter paths, move more often, and move further per displacement than do toads from older (long-established) populations. Are these higher movement rates of invasion-front toads associated with modified locomotor performance (e.g. speed, endurance)? In an outdoor raceway, toads collected from the invasion front had similar speeds, but threefold greater endurance, compared to conspecifics collected from a long-established population. Thus, increased daily displacement in invasion-front toads does not appear to be driven by changes in locomotor speed. Instead, increased dispersal is associated with higher endurance, suggesting that invasion-front toads tend to spend more time moving than do their less dispersive conspecifics. Whether this increased endurance is a cause or consequence of behavioural shifts associated with rapid dispersal is unclear. Nonetheless, shifts in endurance between frontal and core populations of this invasive species point to the complex panoply of traits affected by selection for increased dispersal ability on expanding population fronts.

Evidence for Predation on Terrestrial Cane Toads 'Bufo Marinus' by the Sooty Grunter 'Hephaestus Fuliginosus' in Northern Australia

Expansion of the Cane Toad Bufo marinus across Australia has been implicated in the population declines of a range of indigenous terrestrial and semi-aquatic vertebrate fauna. Stomach content analysis of a number of Sooty Grunter 'Hephaestus fuliginosus' recently collected in the Northern Territory has highlighted occasional consumption of the terrestrial life history stages of Cane Toads by this widespread northern Australian freshwater fish, with minimal apparent effect. The impacts of Cane Toad invasion on freshwater fish communities remains one of the many information gaps surrounding the ecosystem effects of toad colonisation in Australia.

Vulnerability of an Australian anuran tadpole assemblage to the toxic eggs of the invasive cane toad (Bufo marinus)

The invasion of cane toads (Bufo marinus) across tropical Australia has fatally poisoned many native predators; the most frequent victims may be tadpoles of native frogs, which die when they consume the toxic eggs of the toads. Field studies have documented high and species-specific mortality of tadpoles following toad spawning.To clarify the determinants of tadpole vulnerability, we conducted 1593 laboratory trials in which single tadpoles were exposed to 10 toad eggs, either with or without an alternative food source (lettuce). At least some tadpoles within all 15 species tested consumed toad eggs. Interspecific variance in survival rates (from 0 to >70%) was driven by feeding responses not by physiological tolerance to toxins: almost all native tadpoles that consumed eggs died rapidly.Tadpole mortality was decreased by the presence of an alternative food source in four species, increased in two species, and not affected in seven species. In three of four taxa where we tested both small (early-stage) and large (late-stage) tadpoles, both mean survival rates and the effects of alternative food on survival shifted with tadpole body size. Trials with one species (Limnodynastes convexiusculus) showed no significant inter-clutch variation in feeding responses or tolerance to toxins. Overall, our data show that cane toad eggs are highly toxic to native anuran tadpoles, but that whether or not a tadpole is killed by encountering toad eggs depends upon a complex interaction between the native anuran's species, its body size, and whether or not alternative food was present. In nature, larval vulnerability also depends upon the seasonal timing and location of spawning events, and habitat selection and foraging patterns of the tadpoles. Our results highlight the complexity of vulnerability determinants, and identify ecological factors (rather than physiology or feeding behaviour) as the primary deter- minants of cane toad impact on native tadpoles.

The evolution of growth rates on an expanding range edge

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

Something different for dinner? Responses of a native Australian predator (the keelback snake) to an invasive prey species (the cane toad)

Predictions from foraging theory suggest that the probability a native predator will incorporate a novel type of prey (such as an invasive species) into its diet depends upon the potential benefits (e.g., nutrient input) vs. costs (e.g., handling time) of ingesting it. Cane toads (Bufo marinus) were introduced to Australia in 1935 and are highly toxic to many frog-eating snakes, thus there was strong selection to delete toads from the diet of these species. What has happened, however, to the feeding responses of an Australian snake species that is able to consume toads without dying? Our field surveys in northeastern Queensland show that, despite their high tolerance to toad toxins (compared to other native snakes), keelbacks (Tropidonophis mairii) feed primarily on native frogs rather than cane toads. This pattern occurs because the snakes show active prey preferences; even under standardized conditions in the laboratory, snakes are more likely to consume frogs than toads. When they are force-fed, snakes frequently regurgitate toads but not frogs. Thus, despite the high availability of the abundant toads, these invasive anurans are largely avoided as prey. This probably occurs because consumption of toads, although not lethal to keelbacks, causes significant sublethal effects and confers little nutritional benefit. Hence, keelback populations are not threatened by toad invasion, but neither do the snakes benefit substantially from the availability of a new type of potential prey.

Maladaptive traits in invasive species: in Australia, cane toads are more vulnerable to predatory ants than are native frogs

1. If a species is translocated outside its native range, some of its traits (evolved to match conditions in the ancestral range) likely will be maladaptive. Identifying ways in which the invader are poorly suited to its new range might provide novel opportunities for biocontrol. 2. The spread of cane toads (Bufo marinus, native to central and South America) through tropical Australia has created major ecological problems. Although many native predators cannot deal with the toxins of the invasive toads, 'meat ants' (Iridomyrmex reburrus) kill and consume many metamorph toads. Might this be a mismatch between the invader and its newly invaded range, whereby the morphology, locomotor ability and/or behaviour of cane toads renders them vulnerable to a predator that poses little danger to native anurans? 3. To explore this possibility, we measured habitat use and activity patterns in meat ants, metamorph cane toads and metamorphs of seven native frog species (Litoria bicolor, L. caerulea, L. dahlii, L. nasuta, L. rothii, Limnodynastes convexiusculus, Opisthodon ornatus) in standardized enclosures in the laboratory. 4. Unlike the frogs, (1) toads selected open microhabitats and were active diurnally, thus increasing encounter rates with meat ants; (2) toads failed to detect and evade approaching ants; (3) toads exhibited poor locomotor ability (short slow hops, reflecting their small size and short limbs); and (4) toads frequently relied on an ineffective defence mechanism (crypsis) when attacked. 5. In combination, these traits rendered cane toad metamorphs far more susceptible to predation by meat ants than were any of the native frogs tested. That vulnerability presumably reflects lack of coevolution between cane toads and Australian ants. 6. The inability of invasive toads to escape predatory native ants might be exploited to reduce cane toad numbers, by manipulating ant densities and/or locations during periods of toad metamorph emergence.

Fatal attraction: adaptations to prey on native frogs imperil snakes after invasion of toxic toads

Adaptations that enhance fitness in one situation can become liabilities if circumstances change. In tropical Australia, native snake species are vulnerable to the invasion of toxic cane toads. Death adders (Acanthophis praelongus) are ambush foragers that (i) attract vertebrate prey by caudal luring and (ii) handle anuran prey by killing the frog then waiting until the frog's chemical defences degrade before ingesting it. These tactics render death adders vulnerable to toxic cane toads (Bufo marinus), because toads elicit caudal luring more effectively than do native frogs, and are more readily attracted to the lure. Moreover, the strategy of delaying ingestion of a toad after the strike does not prevent fatal poisoning, because toad toxins (unlike those of native frogs) do not degrade shortly after the prey dies. In our laboratory and field trials, half of the death adders died after ingesting a toad, showing that the specialized predatory behaviours death adders use to capture and process prey render them vulnerable to this novel prey type. The toads' strong response to caudal luring also renders them less fit than native anurans (which largely ignored the lure): all toads bitten by adders died. Together, these results illustrate the dissonance in behavioural adaptations that can arise following the arrival of invasive species, and reveal the strong selection that occurs when mutually naive species first interact.

Native Australian frogs avoid the scent of invasive cane toads

AbstractInvasive species can affect the ecosystems they colonize by modifying the behaviour of native taxa; for example, avoidance of chemical cues from the invader may modify habitat use (shelter site selection) by native species. In laboratory trials, we show that metamorphs of most (but not all) native frog species on a tropical Australian floodplain avoid the scent of invasive cane toads (Bufo marinus Linnaeus 1758). Cane toads also avoid conspecific scent. This response might reduce vulnerability of metamorph frogs and toads to larger predatory toads. However, similar avoidance of one type of pungency control (garlic), and the presence of this avoidance behaviour in frogs at the toad invasion front (and hence, with no prior exposure to toads), suggest that this may not be an evolved toad‐specific response. Instead, our data support the simpler hypothesis that the metamorph anurans tend to avoid shelter sites that contain strong and unfamiliar scents. Temporal and spatial differences in activity of frogs versus toads, plus the abundance of suitable retreat sites during the wet season (the primary time of frog activity), suggest that avoiding toad scent will have only a minor impact on the behaviour of native frogs. However, this behavioural impact may be important when environmental conditions bring toads and frogs into closer contact.

Comparisons through time and space suggest rapid evolution of dispersal behaviour in an invasive species

During a biological invasion, we expect that the expanding front will increasingly become dominated by individuals with better dispersal abilities. Over many generations, selection at the invasion front thus will favour traits that increase dispersal rates. As a result of this process, cane toads (Bufo marinus) are now spreading through tropical Australia about 5-fold faster than in the early years of toad invasion; but how have toads changed to make this happen? Here we present data from radio-tracking of free-ranging cane toads from three populations (spanning a 15-year period of the toads’ Australian invasion, and across 1800 km). Our data reveal dramatic shifts in behavioural traits (proportion of nights when toads move from their existing retreat-site to a new one, and distance between those successive retreat-sites) associated with the rapid acceleration of toad invasion. Over a maximum period of 70 years (~50 generations), cane toads at the invasion front in Australia apparently have evolved such that populations include a higher proportion of individuals that make long, straight moves.

Impact of the invasive cane toad (Bufo marinus) on an Australian frog (Opisthodon ornatus) depends on minor variation in reproductive timing

Invasive species are widely viewed as unmitigated ecological catastrophes, but the reality is more complex. Theoretically, invasive species could have negligible or even positive effects if they sufficiently reduce the intensity of processes regulating native populations. Understanding such mechanisms is crucial to predicting ultimate ecological impacts. We used a mesocosm experiment to quantify the impact of eggs and larvae of the introduced cane toad (Bufo marinus) on fitness-related traits (number, size and time of emergence of metamorphs) of a native Australian frog species (Opisthodon ornatus). The results depended upon the timing of oviposition of the two taxa, and hence the life-history stages that came into contact. Growth and survival of O. ornatus tadpoles were enhanced when they preceded B. marinus tadpoles into ponds, and reduced when they followed B. marinus tadpoles into ponds, relative to when tadpoles of both species were added to ponds simultaneously. The dominant tadpole-tadpole interaction is competition, and the results are consistent with competitive priority effects. However, these priority effects were reduced or reversed when O. ornatus tadpoles encountered B. marinus eggs. Predation on toxic toad eggs reduced the survival of O. ornatus and B. marinus. The consequent reduction in tadpole densities allowed the remaining O. ornatus tadpoles to grow more rapidly and to metamorphose at larger body sizes (>60% disparity in mean mass). Thus, exposure to B. marinus eggs reduced the number of O. ornatus metamorphs, but increased their body sizes. If the increased size at metamorphosis more than compensates for the reduced survival, the effective reproductive output of native anurans may be increased rather than decreased by the invasive toad. Minor interspecific differences in the seasonal timing of oviposition thus have the potential to massively alter the impact of invasive cane toads on native anurans.

A native dasyurid predator (common planigale, Planigale maculata) rapidly learns to avoid a toxic invader

Abstract Interactions between invasive species and native fauna afford a unique opportunity to examine interspecific encounters as they first occur, without the complications introduced by coevolution. In northern Australia, the continuing invasion of the highly toxic cane toad Bufo marinus poses a threat to many frog‐eating predators. Can predators learn to distinguish the novel toxic prey item from native prey (and thus, avoid being poisoned), or are longer‐term genetically based changes to attack behaviour needed before predators can coexist with toads? To predict the short‐term impact of cane toads on native predators, we need to know the proportion of individuals that will attack toads, the proportion surviving the encounter, and whether surviving predators learn to avoid toads. We quantified these traits in a dasyurid (common planigale, Planigale maculata) that inhabits tropical floodplains across northern Australia. Although 90% of naïve planigales attacked cane toads, 83% of these animals survived because they either rejected the toad unharmed, or killed and consumed the prey snout‐first (thereby avoiding the toxin‐laden parotoid glands). Most planigales showed one‐trial learning and subsequently refused to attack cane toads for long time periods (up to 28 days). Toad‐exposed planigales also avoided native frogs for up to 9 days, thereby providing an immediate benefit to native anurans. However, the predators gradually learnt to use chemical cues to discriminate between frogs and toads. Collectively, our results suggest that generalist predators can learn to distinguish and avoid novel toxic prey very rapidly – and hence, that small dasyurid predators can rapidly adapt to the cane toad invasion. Indeed, it may be feasible to teach especially vulnerable predators to avoid cane toads before the toads invade, by deploying low‐toxicity baits that stimulate taste‐aversion learning.

Mass mortality of native anuran tadpoles in tropical Australia due to the invasive cane toad ( Bufo marinus)

Specific pathways of the ecological impact of invasive species remain poorly known. Although the spread of toxic cane toads ( Bufo marinus) through tropical Australia is widely believed to have caused extensive mortality of native reptiles and mammals, effects of toad ingestion on native anurans have been virtually ignored. Our studies on the Adelaide River floodplain show that the most numerous vertebrate victims of toad invasion are native tadpoles that die when they attempt to consume toad eggs. We documented 11 episodes of mass mortality, totalling >1300 tadpoles of 10 species, in five waterbodies within a single wet-season shortly after the toads invaded. A causal link between toad breeding and tadpole mortality is supported by observations that: (1) in at least 9 of the 11 waterbodies involved, toads bred immediately prior to mortality events; (2) water quality was indistinguishable from that of control ponds, and tadpoles placed in that water remained healthy; (3) dead tadpoles showed no sign of disease; and (4) laboratory trials showed rapid, 100% mortality in native tadpoles exposed to freshly-laid toad eggs. Despite these high mortality rates, toad invasion does not appear to threaten the viability of anuran populations because frogs often breed in ponds not used by toads, and because density-dependent growth and survival within tadpole communities mean that additional mortality may not reduce the total effective recruitment of metamorph frogs from a waterbody.

Invasive cane toads ( Bufo marinus) cause mass mortality of freshwater crocodiles ( Crocodylus johnstoni) in tropical Australia

Invasive species are frequently blamed for faunal declines, but there is little direct evidence about the pathways, magnitude and size-selectivity of mortality induced by invaders. Top predators are of particular interest in this context, because their removal can generate substantial cascades of secondary effects on community composition. Cane toads ( Bufo marinus) are large South American anurans currently spreading rapidly through tropical Australia. Native predators that attempt to consume these highly toxic toads may die as a result. During surveys of the Victoria River in the semi-arid tropical region of the Northern Territory, we documented massive mortality of freshwater crocodiles ( Crocodylus johnstoni) at the toad invasion front. Dead crocodiles spanned a wide size range (0.6–2.1 m long) but with significant biases; intermediate-sized animals (0.6–1.5 m long) were more likely to be found dead. Population densities of crocodiles plummeted by as much as 77% following toad invasion, and population size-structures changed. The negative impacts of toads on crocodiles appear to be greater in these hot semi-arid landscapes than in cooler, higher rainfall areas where crocodiles have access to a wider prey base, and the toads are less prone to desiccation and can rehydrate in small, scattered water bodies rather than in the main river. Hence, the impact of cane toad invasion on this top predator may increase with increasing aridity.

A Toad More Traveled: The Heterogeneous Invasion Dynamics of Cane Toads in Australia

To predict the spread of invasive species, we need to understand the mechanisms that underlie their range expansion. Assuming random diffusion through homogeneous environments, invasions are expected to progress at a constant rate. However, environmental heterogeneity is expected to alter diffusion rates, especially by slowing invasions as populations encounter suboptimal environmental conditions. Here, we examine how environmental and landscape factors affect the local invasion speeds of cane toads (Chaunus [Bufo] marinus) in Australia. Using high-resolution cane toad data, we demonstrate heterogeneous regional invasion dynamics that include both decelerating and accelerating range expansions. Toad invasion speed increased in regions characterized by high temperatures, heterogeneous topography, low elevations, dense road networks, and high patch connectivity. Regional increases in the toad invasion rate might be caused by environmental conditions that facilitate toad reproduction and movement, by the evolution of long-distance dispersal ability, or by some combination of these factors. In any case, theoretical predictions that neglect environmental influences on dispersal at multiple spatial scales may prove to be inaccurate. Early predictions of cane toad range expansion rates that assumed constant diffusion across homogeneous landscapes already have been proved wrong. Future attempts to predict range dynamics for invasive species should consider heterogeneity in (1) the environmental factors that determine dispersal rates and (2) the mobility of invasive populations because dispersal-relevant traits can evolve in exotic habitats. As an invasive species spreads, it is likely to encounter conditions that influence dispersal rates via one or both of these mechanisms.