Risk level of chemical cues determines retention of recognition of new predators in Iberian green frog tadpoles

In aquatic environments, many prey rely on chemosensory information from injured (alarm cues) or stressed conspecifics (disturbance cues) to assess predation risk. Alarm cues are considered as a sign of higher risk than disturbance cues. These cues could be used by prey to learn potential new predators. In this study, we tested whether Iberian green frog tadpoles (Pelophylax perezi) exhibited antipredator responses to alarm and disturbance cues of conspecifics and whether tadpoles could associate new predators with alarm or disturbance cues. Tadpoles reduced their activity in the presence of disturbance cues, but only weakly when compared with their response to alarm cues. Also, tadpoles learned to recognize new predators from association with alarm or disturbance cues. However, the period of retention of the learned association was shorter for disturbance than alarm cues. Our results indicate that tadpoles are able to modify their antipredatory behavior according to (1) the degree of risk implied by the experimental cues (2) their previous experience of chemical cues of the predator.     

Different antipredator behaviour in two anuran tadpoles: effects of predator diet

Recent investigations have indicated that animals are able to use chemical cues of predators to assess the magnitude of predation risk. One possible source of such cues is predator diet. Chemical cues may also be important in the development of antipredator behaviour, especially in animals that possess chemical alarm substances. Tadpoles of the common toad (Bufo bufo) are unpalatable to most vertebrate predators and have an alarm substance. Tadpoles of the common frog (Rana temporaria) lack both these characters. We experimentally studied how predator diet, previous experience of predators and body size affect antipredator behaviour in these two tadpole species. Late-instar larvae of the dragonfly Aeshna juncea were used as predators. The dragonfly larvae were fed a diet exclusively of insects, R. temporaria tadpoles or B. bufo tadpoles. R. temporaria tadpoles modified their behaviour according to the perceived predation risk. Depending on predator diet, the tadpoles responded with weak antipredatory behaviour (triggered by insect-fed predators) or strong behaviour (triggered by tadpole-fed predators) with distinct spatial avoidance and lowered activity level. The behaviour of B. bufo in predator diet treatments was indistinguishable from that in the control treatment. This lack of antipredator behaviour is probably related to the effective post-encounter defenses and more intense competitive regime experienced by B. bufo. The behaviour of both tadpole species was dependent on body size, but this was not related to predator treatments. Our results also indicate that antipredator behaviour is largely innate in tadpoles of both species and is not modified by a brief exposure to predators. Received: 22 August 1996 / Accepted after revision: 31 January 1997     

Chemical communication in green and golden bell frogs: do tadpoles respond to chemical cues from dead conspecifics?

Captive bred animals often lack the ability of predator recognition and predation is one of the strongest causes of failure of breed and release projects. Several tadpole and fish species respond defensively to chemical cues from injured or dead conspecifics, often referred to as alarm pheromones. In natural conditions and in species that school, the association of chemical cues from predators to alarm pheromones released by attacked conspecifics may lead to the learning of the predator-related danger without experiencing an attack. In the laboratory, this chemical communication can also be used in associative learning techniques to teach naïve tadpoles to avoid specific predators and improve survivorship of released animals. In our experimental trials, tadpoles of the threatened green and golden bell frog (Litoria aurea) did not avoid or decrease their activity when exposed to solutions of conspecific macerate, suggesting that the chemicals released into the water by dead/injured conspecifics do not function as an alarm pheromone. This non-avoidance of dead conspecific chemicals may explain why green and golden bell frog tadpoles have seemingly not developed any avoidance behaviour to the presence of introduced mosquito fish, and may render attempts to teach naïve tadpoles to avoid this novel predator more difficult.     

Predation risk assessment by green frog (<Emphasis Type="Italic">Rana clamitans</Emphasis>) tadpoles through chemical cues produced by multiple prey

Many prey assess predation risk through predator chemical cues. Numerous studies have shown that (1) prey sometimes respond to chemical cues produced by heterospecifics and (2) that many species are capable of associative learning. This study extends this research by focusing on predation risk assessment and antipredator behavior in environments containing chemical cues produced by multiple prey species. The results show that green frog (Rana clamitans) tadpoles (1) assess risk from the chemical cue produced during predation by a heterospecific (gray tree frog, Hyla versicolor, tadpoles) and (2) can exhibit similarly strong behavioral responses to a mix of conspecific and heterospecific cues compared to conspecific cue alone, depending on their conditioning environment. I then discuss how the prey choice of the predators and the relative abundances of the prey species should influence the informational value of heterospecific cues.     

Degradation of chemical alarm cues under natural conditions: risk assessment by larval woodfrogs

Summary. Many aquatic species use chemosensory information to assess predation risk. The cues used in such risk assessment can come either from the predator (predator odour) or from injured prey (alarm cues). The information conveyed through chemicals may, however, be inaccurate both spatially and temporally, as chemicals may persist in the environment long after the predator is gone. Thus, the level of accuracy of the cues for risk assessment may depend on the persistency of the chemicals in the habitat. Here, we investigated the persistency of alarm cues of a larval amphibian, the woodfrog (Rana sylvatica) in a ephemeral pond, their natural habitat. We introduced either alarm cues or control water in enclosed sleeves (~10 L) installed in the pond. The sleeve water was then sampled after 5 min and every two hours for eight hours. We used the behavioural response of woodfrog tadpoles to alarm cues as a bioassay to assess how long the alarm cues persisted in the environment. We found that tadpoles responded with an antipredator response to the pond water containing alarm cues 5 min after the injection of the cues in the sleeves but did not respond to that same pond water after two hours. Our results indicate that biodegradation and/or photodegradation of alarm cues in natural habitats might occur relatively quickly as the loss of a response to the cues in our experiment was independent of a dilution effect. This contrasts with previous laboratory results indicating that chemicals may be active after several hours.     

The paradox of risk assessment: comparing responses of fathead minnows to capture-released and diet-released alarm cues from two different predators

Summary. Many aquatic prey are known to use chemical alarm cues to assess their risk of predation. In fishes, such alarm cues can be released either through damage of the epidermis during a predatory attack (capture-released) or through release from the predator feces (diet-released). In our study, we compared the importance of capture- versus diet-released alarm cues in risk assessment by fathead minnows (Pimephales promelas) that were na?ve to fish predators. We utilized two different fish predators: a specialized piscivore, the northern pike (Esox lucius) and a generalist predator, the brook trout (Salvelinus fontinalis). Handling time of pike consuming minnows was much shorter than for trout consuming minnows, likely resulting in less epidermal damage to the minnows during attacks by pike. In accordance with this, minnows showed a less intense antipredator response to capture-released cues from pike than capture-released cues from trout. This represents a paradox in risk assessment for the minnows as they respond to the specialized piscivore, the more dangerous predator, with a less intense antipredator response. In contrast, the minnows showed a stronger antipredator response to the specialized piscivore than to the generalist when given diet cues. This work highlights the need for researchers to carefully consider the nature of the information available to prey in risk assessment.     

Antipredator defenses along a latitudinal gradient in Rana temporaria

Antipredator defenses are expected to decrease toward higher latitudes because predation rates are predicted to decrease with latitude. However, latitudinal variation in predator avoidance and defense mechanisms has seldom been studied. We studied tadpole antipredator defenses in seven Rana temporaria populations collected along a 1500-km latitudinal gradient across Sweden, along which previous studies have found increasing tadpole growth and development rates. In a laboratory common garden experiment, we measured behavioral and morphological defenses by raising tadpoles in the presence and absence of a predator (Aeshna dragonfly larva) in two temperature treatments. We also estimated tadpole survival in the presence of free-ranging predators and compared predator densities between R. temporaria breeding ponds situated at low and high latitudes. Activity and foraging were generally positively correlated with latitude in the common garden experiment. While all populations responded to predator presence by decreasing activity and foraging, high-latitude populations maintained higher activity levels in the presence of the predator. All populations exhibited defensive morphology in body and tail shape. However, whereas tail depth tended to increase with latitude in the presence of predator, it did not change with latitude in the absence of the predator. Predator presence generally increased larval period and decreased growth rate. In the southern populations, predator presence tended to have a negative effect on metamorphic size, whereas in the northern populations predators had little or a positive effect on size. Latitude of origin had a strong effect on survival in the presence of a free-ranging predator, with high-latitude tadpoles experiencing higher mortality than those from the low latitudes. In the wild, predator densities were significantly lower in high-latitude than in mid-latitude breeding ponds. Although the higher activity level in the northern populations seems to confer a significant survival disadvantage under predation risk, it is probably needed to maintain the high growth and development rates. However, the occurrence of R. temporaria at high latitudes may be facilitated by the lower predator densities in the north.     

A review of predator diet effects on prey defensive responses

Numerous studies have examined how predator diets influence prey responses to predation risk, but the role predator diet plays in modulating prey responses remains equivocal. We reviewed 405 predator–prey studies in 109 published articles that investigated changes in prey responses when predators consumed different prey items. In 54 % of reviewed studies, prey responses were influenced by predator diet. The value of responding based on a predator’s recent diet increased when predators specialized more strongly on particular prey species, which may create patterns in diet cue use among prey depending upon whether they are preyed upon by generalist or specialist predators. Further, prey can alleviate costs or accrue greater benefits using diet cues as secondary sources of information to fine tune responses to predators and to learn novel risk cues from exotic predators or alarm cues from sympatric prey species. However, the ability to draw broad conclusions regarding use of predator diet cues by prey was limited by a lack of research identifying molecular structures of the chemicals that mediate these interactions. Conclusions are also limited by a narrow research focus. Seventy percent of reviewed studies were performed in freshwater systems, with a limited range of model predator–prey systems, and 98 % of reviewed studies were performed in laboratory settings. Besides identifying the molecules prey use to detect predators, future studies should strive to manipulate different aspects of prey responses to predator diet across a broader range of predator–prey species, particularly in marine and terrestrial systems, and to expand studies into the field.     

Opposite shifts in size at metamorphosis in response to larval and metamorph predators

Predation risk can cause organisms to alter the timing of life history switch points. Theory suggests that increased risk in an early life stage should select for switching earlier and smaller, while increased risk in the subsequent stage should select for switching later and larger. This framework has frequently been applied to metamorphosis in amphibians, with mixed results. Few studies examining the effect of larval predation risk on metamorphosis have observed the predicted pattern, and no studies, to our knowledge, have examined the effect of increased risk during and after metamorphosis on the timing of this switch point. Here we examine the effect of larval and post-metamorphic predation risk on metamorphosis in the red-eyed treefrog, Agalychnis callidryas. We raised tadpoles in the presence or absence of cues from caged water bugs fed larvae and cues from spiders fed emerging metamorphs. Water bugs are effective larval predators, while spiders are poor larval predators but prey on metamorphs. Furthermore, since spiders forage on the water surface it is possible that tadpoles could assess future risk from this predator. Predators induced opposite shifts in life history. Tadpoles emerged smaller and less developed in response to water bugs, but later and larger in response to spiders. Interestingly, predator effects on larval duration were not independent; tadpoles delayed emerging in response to spiders, but only in the absence of water bugs.     

To beg or to freeze: multimodal sensory integration directs behavior in a tadpole

Effective coordination of behaviors such as foraging and avoiding predators requires an assessment of cues provided by other organisms. Integrating cues from multiple sensory modalities may enhance the assessment. We studied cue integration by tadpoles of Oophaga pumilio, which live in small arboreal water pools. In this species, mothers periodically visit their tadpoles and feed them with unfertilized eggs. When mothers visit, tadpoles beg conspicuously by vibrating until fed. However, animals other than mother frogs including potential predators may visit water pools. Thus, when a visitor appears, tadpoles must use visitor cues to decide whether to beg or to remain inactive to avoid predation. To elucidate the cues that prompt these behaviors, we videotaped behavior of O. pumilio tadpoles in response to isolated and multimodal cues. Tadpoles swam more when exposed to visual or visual and chemical cues of adult O. pumilio but only exhibited begging when exposed to visual, chemical, and tactile cues together. Visual, chemical, and tactile cues from either male or female adult O. pumilio stimulated swimming and begging, but the same cues from similarly sized heterospecific frogs did not. Lastly, tadpoles exposed to a potential predator did not beg and swam less than tadpoles with no stimulus. Together, these findings suggest that O. pumilio tadpoles use multimodal cues to modulate swimming behavior accordingly in the presence of egg provisioners, predators, and other visitors and that tadpole begging is induced by multimodal cues of conspecific frogs such that tactile and perhaps chemical cues supplement visual cues.     

Survival trade-offs between two predator-induced phenotypes in Pacific treefrogs (Pseudacris regilla)

In many organisms, specific predator species induce defensive phenotypes that are qualitatively different from the phenotypes induced by other predator species. This differential induction implies that there is no optimal phenotype that works best against all predators. However, few studies have actually tested the hypothesis that each predator-induced phenotype provides the highest survival rate in encounters with the predator that induced that phenotype. In this experiment, I reared Pacific treefrog (Pseudacris regilla) larvae with chemical cues from two different predators (bluegill sunfish and predaceous diving-beetle larvae), and without predator cues. The Pacific treefrog larvae in the three treatments differed in their morphology and foraging behavior. I then exposed tadpoles from each treatment to free-foraging predaceous diving beetles and bluegill sunfish. Tadpoles survived best when exposed to the predator whose cues they were reared with, and worst when exposed to the other predator. In both predator environments, the tadpoles reared in the nonpredator control treatment had intermediate survival between the two predator-induced groups. Thus, there is no generalized "antipredator" response to these predators; rather, there was a clear trade-off in survival abilities between the predators.     

Sensory complementation and the acquisition of predator recognition by salmonid fishes

Following disturbance, some aquatic prey species release chemicals that act as a warning cue and increase vigilance in nearby conspecifics. Such disturbance cues evoke consistent low intensity anti-predator responses. In contrast, alarm cues from injured conspecifics often evoke stronger intensity responses in prey animals. In this study, we test the sensory complement hypothesis, which suggests that multiple cues act in an additive or synergistic fashion to provide additional information for risk assessment by prey. In the first experiment, we showed that juvenile rainbow trout pre-exposed to disturbance cues respond to a given concentration of damage-released alarm cues with a higher intensity of response than the trout that were pre-exposed to cues from undisturbed conspecifics. The two cues acted in an additive fashion. In the second experiment, we demonstrated that disturbance cues alone were not enough to elicit a conditioned response to the odour of a novel predator. We also showed that while disturbance cues elicit an increase in the response of trout to alarm cues, this increase does not translate into a stronger learned response to the predator when the predator odour is paired with alarm cues. Future studies should take into account sensory complementation to avoid underestimating the responses of prey to predators.     

Effects of embryonic exposure to conspecific chemical cues on hatching and larval traits in the common frog <Emphasis Type="Italic">(Rana temporaria)</Emphasis>

Summary. Recent studies indicate that amphibian eggs are capable of hatching plasticity in response to chemical cues released by predators feeding on conspecific eggs or larvae. However, information is scarce on the relative importance of predator and conspecific cues in such a process. In particular, no attempt has been made to compare the effects of embryonic exposures to chemical cues indicative of a predation risk for eggs and larvae, although both life stages can co-occur in natural habitats. In this context, common frog embryos (Rana temporaria) were raised until hatching in the presence of crushed conspecific extracts from eggs and tadpoles to assess their respective influences on some hatching and larval traits. While a significant delay in hatching time was observed in embryos exposed to chemical cues from tadpole extract, this life-history shift appeared unaffected by embryonic exposure to egg extract. Hatchlings derived from eggs incubated in the presence of both conspecific extracts showed a significantly greater weight than unexposed controls. However, such an effect was no longer apparent 15, 30 and 50 days after hatching, suggesting that embryonic exposure to chemical cues from damaged conspecific eggs and tadpoles has no influence on larval growth. Lastly, morphological measurements performed on hatchlings and older tadpoles (15, 30 and 50 days old) revealed no significant effect of embryonic treatments on the shape of body and tail.     

Mechanism of predator–prey detection and behavioral responses in some anuran tadpoles

Predator–prey relationship was studied in three sympatric species of anuran tadpoles. The study design consisted of allowing predaceous Hoplobatrachus tigerinus tadpoles to devour prey tadpoles (Sphaerotheca breviceps and Bufo melanostictus) placed in a plastic tub (five tadpoles of each species, stage ~27) in 30 min. In trials without refugia, more tadpoles of Bufo fell prey compared to Sphaerotheca. In contrast, provision of refugia using hydrilla plant reversed predation risk of the two species. The swimming speed (V _max = 64.55 ± 1.45 cm/s) of Hoplobatrachus tadpoles was much higher compared to the prey species (Bufo: 3.6 ± 0.4 cm/s; Sphaerotheca: 27.6 ± 1.6 cm/s). Poor swimming ability may account for the observed vulnerability of the Bufo tadpoles to predation especially in clear waters; refugia overcame predation to some extent. On the other hand, Sphaerotheca tadpoles that swim faster than the toad tadpoles were less vulnerable in open areas; refugia actually hindered swimming and increased predation. Experiments with association choice tests show that predaceous tadpoles detect prey based on both visual and chemical cues. On the other hand, the prey tadpoles detected predator based exclusively on chemical rather than visual cues. The antipredator defense strategy of the toad tadpoles is manifested in the form of reduced movements, remaining still for longer times and, increased burst speed. The present findings also suggest that in both prey species predator detection has a genetic basis since naive tadpoles with no prior exposure to predators exhibit fright response on first encounter with them.     

The ghost of predation future: threat-sensitive and temporal assessment of risk by embryonic woodfrogs

Amphibians are able to learn to recognize their future predators during their embryonic development (the ghost of predation future). Here, we investigate whether amphibian embryos can also acquire additional information about their future predators, such as the level of threat associated with them and the time of day at which they would be the most dangerous. We exposed woodfrog embryos (Rana sylvatica) to different concentrations of injured tadpole cues paired with the odor of a tiger salamander (Ambystoma tigrinum) between 1500 and 1700 hours for five consecutive days and raised them for 9 days after hatching. First, we showed that embryos exposed to predator odor paired with increasing concentrations of injured cues during their embryonic development subsequently display stronger antipredator responses to the salamander as tadpoles, thereby demonstrating threat-sensitive learning by embryonic amphibians. Second, we showed that the learned responses of tadpoles were stronger when the tadpoles were exposed to salamander odor between 1500 and 1700 hours, the time at which the embryos were exposed to the salamander, than during earlier (1100–1300 hours) or later (1900–2100 hours) periods. Our results highlight the amazing sophistication of learned predator recognition by prey and emphasize the importance of temporal considerations in experiments examining risk assessment by prey.     

Ontogenetic differences in chemical alarm cue production determine antipredator responses and learned predator recognition

How individuals assess, respond and subsequently learn from alarm cues is crucial to their survival and future fitness. Yet this information is not constant through time; many individuals are exposed to different predators throughout their life as they outgrow some predators or move to habitats containing different predators. To maximise overall fitness, individuals should discriminate between different cues and respond and learn from only those that are relevant to their current ontogenetic stage. We tested whether juvenile spiny chromis, Acanthochromis polyacanthus, could distinguish between chemical alarm cues from conspecific donors of different ontogenetic stages and whether the cue ontogenetic stage of the cue donor affected the efficacy of learning about predators. Juveniles displayed a significant antipredator response when conditioned with juvenile chemical alarm cues paired with predator odour but failed to respond when conditioned with predator odour paired with either adult alarm cues or with saltwater. Subsequently, individuals only recognised the predator odour alone as a threat when conditioned with juvenile alarm cues. This demonstrates that prey may be highly specific in how they use information from conspecific alarm cues, selectively responding to and learning from only those cues that are relevant to their developmental stage.     

Threat-sensitive learning of predators by larval mosquitoes <Emphasis Type="Italic">Culex restuans</Emphasis>

A prerequisite for prey to show adaptive behavioural responses to predators is that the prey has the ability to recognise predators as threats. While predator recognition can be innate in many situations, learning is often essential. For many aquatic species, one common way to learn about predators is through the pairing of a novel predator odour with alarm cues released from injured conspecifics. One study with fish demonstrated that this mode of learning not only allows the prey to recognise the predatory cues as a threat, but also mediates the level of threat associated with the predator cues (i.e. threat-sensitive learning). When the prey is exposed to the novel predator with a high concentration of alarm cues, they subsequently show a high intensity of antipredator response to the predator cues alone. When exposed to the predator with a low concentration of alarm cues, they subsequently show a low-intensity response to the predator cues. Here, we investigated whether larval mosquitoes Culex restuans have the ability to learn to recognise salamanders as a threat through a single pairing of alarm cues and salamander odour and also whether they would learn to respond to salamander cues in a threat-sensitive manner. We conditioned individual mosquitoes with water or a low, medium or high concentration of crushed conspecific cues (alarm cues) paired with salamander odour. Mosquitoes exposed to salamander odour paired with alarm cues and subsequently exposed to salamander odour alone responded to the salamander as a threat. Moreover, the intensity of antipredator response displayed during the conditioning phase matched the response intensity during the testing phase. This is the first demonstration of threat-sensitive learning in an aquatic invertebrate.     

Variable predation risk and the dynamic nature of mosquito antipredator responses to chemical alarm cues

Summary. Predation is a pervasive selective agent highly variable in space and time. Due to the costs associated with antipredator responses, prey would be at a selective advantage if they respond to predation threats with an intensitfy matching the threat posed by the predator. Many aquatic organisms have been shown to use chemical alarm cues present in the water to assess the level of risk in their environment. This includes mosquito larvae which show antipredator responses to conspecific alarm cues. In this study, we investigated the nature of the responses of larval mosquitoes Culex restuans to those cues. In our initial observations, we showed pond/population differences in the response intensity of C. restuans to alarm cues. In experiment 1, we showed that the response intensity to alarm cues could be increased by increasing the background level of risk in the mosquitoes’ environment (by adding salamander predators) and once turned on, the response intensity to alarm cues was likely maintained for the remainder of the mosquitoes’ aquatic life. In experiments 2 and 3, we investigated if the increase in response intensity to alarm cues was directly correlated with the level of background risk in the mosquitoes’ environment. When given increasing levels of background risk, mosquito larvae subsequently showed a graded response to conspecific alarm cues. This series of experiments demonstrates that the response intensity of larval mosquitoes to a standard concentration of alarm cues is not fixed, but rather dependent on the background level of risk in the environment. An understanding of the background level of risk is particularly important for comparing antipredator responses of prey between habitats.     

Predator identity and additive effects in a treehole community

Multiple predator species can interact as well as strongly affect lower trophic levels, resulting in complex, nonadditive effects on prey populations and community structure. Studies of aquatic systems have shown that interactive effects of predators on prey are not necessarily predictable from the direct effects of each species alone. To test for complex interactions, the individual and combined effects of a top and intermediate predator on larvae of native and invasive mosquito prey were examined in artificial analogues of water-filled treeholes. The combined effects of the two predators were accurately predicted from single predator treatments by a multiplicative risk model, indicating additivity. Overall survivorship of both prey species decreased greatly in the presence of the top predator Toxorhynchites rutilus. By itself, the intermediate predator Corethrella appendiculata increased survivorship of the native prey species Ochlerotatus triseriatus and decreased survivorship of the invasive prey species Aedes albopictus relative to treatments without predators. Intraguild predation did not occur until alternative prey numbers had been reduced by approximately one-half. Owing to changes in size structure accompanying its growth, T. rutilus consumed more prey as time progressed, whereas C. appendiculata consumed less. The intermediate predator, C. appendiculata, changed species composition by preferentially consuming A. albopictus, while the top predator, T. rutilus, reduced prey density, regardless of species. Although species interactions were in most cases predicted from pairwise interactions, risk reduction from predator interference occurred when C. appendiculata densities were increased and when the predators were similarly sized.     

The avoidance response of fathead minnows to chemical alarm cues: understanding the effects of donor gender and breeding condition

Summary. All animals are vulnerable to predation at some point in their lives and consequently prey organisms often develop effective risk assessment systems. For many aquatic species predation risk assessment occurs through the use of olfactory cues, including predator odours and alarm cues from damaged or disturbed conspecifics. When aquatic species encounter conspecific alarm cues they may respond, or not, based on specific information including cue concentration, health and size of the conspecific donor and potentially the gender and breeding condition of the donor. Previous laboratory studies have demonstrated that fathead minnows (Pimephales promelas) fail to respond to the skin extracts of breeding male minnows. The purpose of the current study was to verify these early laboratory findings in the field as well as to further investigate the effect of female reproductive state and donor gender on the response of minnows to damage-release alarm cues. Our results indicate that male breeding condition has a significant effect on how minnows will respond to conspecific cues. Minnows showed avoidance of cues of female minnows and male minnows not in breeding condition, in comparison to cues of breeding male minnows and cues of male and female swordtails. Neither the gender of non-breeding minnows nor the reproductive state of female minnows influenced the avoidance of minnows to alarm cues.