Conspecific Chemical Alarm Cues Research Articles

Behavioral response to chemical cues from injured conspecifics in the livebearing fish, Brachyrhaphis rhabdophora

AbstractPredator–prey dynamics have led to a strong selection of prey's ability to detect and respond to information about the risk environment. Further, intrinsic factors, such as sex, may cause prey to perceive and respond to information differently. Chemical alarm cues from injured conspecifics are a classic example of how prey have evolved to use publicly available information to shape their behavior and enhance fitness, yet sex‐specific alarm reactions are rarely considered. The purpose of our study was to compare how males and females respond to conspecific chemical alarm cues in the livebearing fish species, Brachyrhaphis rhabdophora. Furthermore, we tested males and females from populations with a high‐ or low‐predation environment. Brachyrhaphis rhabdophora showed strong alarm reactions, but contrary to our predictions, showed limited variation due to sex or predation environment. We found that males and females from both populations displayed lower activity levels and increased their swimming depth when exposed to an alarm cue, despite variable and consistent baseline behaviors among individuals. These data further contribute to our understanding of what factors shape the evolution of behavioral responses to chemical alarm cues in fishes.

Acquired predator recognition via epidermal alarm cues but not dietary alarm cues by isolated pupfish.

We tested whether Shoshone pupfish Cyprinodon nevadensis shoshone and Amargosa River pupfish C. n. amargosae respond behaviourally to conspecific chemical alarm cues released when epidermal tissue is damaged by a predator. We found that both subspecies reduced activity and vertical position in the water column in response to alarm cues. We then tested if pupfish can use alarm cue to acquire recognition of a novel predator. We trained pupfish with (1) water + odour of largemouth bass fed a diet of earthworms, (2) alarm cues from skin extract (epidermal alarm cues) + odour of bass fed a diet of earthworms, or (3) water + odour of bass fed a diet of pupfish (dietary alarm cues). Pupfish responded to epidermal alarm cues but not to dietary alarm cues. Pupfish were retested with the odour of bass that were fed an earthworm diet. Pupfish that had previously received epidermal alarm cues reduced vertical position and activity relative to the other two treatments. This is the first demonstration of acquired recognition of a novel predator by a pupfish, the first report of partial predator naiveté, and opens the possibility of predator-recognition training as a tool for management and conservation of endangered desert fishes.

Consistent Antipredator Behavioral Responses among Populations of Red River Pupfish with Disparate Predator Communities

Many desert fishes, which evolved in isolated aquatic “islands” with limited predation pressure, have been severely impacted by non-native predators. These impacts have been attributed to the evolutionary loss of antipredator competence, known as the predator naiveté hypothesis. Recent work provided support for this hypothesis for one desert fish species. We sought to examine the generality of the predator naiveté hypothesis by evaluating antipredator competence in five populations of Red River pupfish (Cyprinodon rubrofluviatilis), a species that occupies habitats that vary in the degree of isolation and levels of fish species richness. Fish were exposed to a conspecific chemical alarm cue released from damaged epidermal tissue as a general assay of antipredator response. We found that pupfish from all five populations exhibited antipredator behavior in response to the alarm cue, regardless of the exposure to predation risk. These data provide evidence that antipredator responses to alarm cues are conserved in Red River pupfish, even in populations isolated from piscivorous species.

Alarm cues and alarmed conspecifics: neural activity during social learning from different cues in Trinidadian guppies.

Learning to respond appropriately to novel dangers is often essential to survival and success, but carries risks. Learning about novel threats from others (social learning) can reduce these risks. Many species, including the Trinidadian guppy (Poecilia reticulata), respond defensively to both conspecific chemical alarm cues and conspecific anti-predator behaviours, and in other fish such social information can lead to a learned aversion to novel threats. However, relatively little is known about the neural substrates underlying social learning and the degree to which different forms of learning share similar neural mechanisms. Here, we explored the neural substrates mediating social learning of novel threats from two different conspecific cues (i.e. social cue-based threat learning). We first demonstrated that guppies rapidly learn about threats paired with either alarm cues or with conspecific threat responses (demonstration). Then, focusing on acquisition rather than recall, we discovered that phospho-S6 expression, a marker of neural activity, was elevated in guppies during learning from alarm cues in the putative homologue of the mammalian lateral septum and the preoptic area. Surprisingly, these changes in neural activity were not observed in fish learning from conspecific demonstration. Together, these results implicate forebrain areas in social learning about threat but raise the possibility that circuits contribute to such learning in a stimulus-specific manner.

Ignorance is not bliss: evolutionary naiveté in an endangered desert fish and implications for conservation.

Predator naiveté has been invoked to explain the impacts of non-native predators on isolated populations that evolved with limited predation. Such impacts have been repeatedly observed for the endangered Pahrump poolfish, Empetrichthys latos, a desert fish species that evolved in isolation since the end of the Pleistocene. We tested Pahrump poolfish anti-predator responses to conspecific chemical alarm cues released from damaged epidermal tissue in terms of fish activity and water column position. Pahrump poolfish behavioural responses to conspecific alarm cues did not differ from responses to a dechlorinated tap water control. As a positive control, the well-studied fathead minnow, Pimephales promelas, showed significant alarm cue responses in terms of reduced activity and lowered water column position. The density of epidermal club cells, the presumptive source of alarm cues, was significantly lower in Pahrump poolfish relative to fathead minnows. Therefore, anti-predator competence mediated by conspecific alarm cues does not seem to be a component of the ecology of Pahrump poolfish. These findings provide a proximate mechanism for the vulnerability of Pahrump poolfish to non-native predators, with implications for the conservation and management of insular species.

Early-life and parental predation risk shape fear acquisition in adult minnows.

Exposure to predation risk can induce a fearful baseline state, as well as fear reactions toward novel situations (i.e., neophobia). Some research indicates that risk exposure during sensitive periods makes adults more prone to acquiring long-term fearful phenotypes. However, chronic risk can also lead to ignoring threats in order to maintain other activities. We sought to assess how a relatively long period of low risk, experienced either early in life or by the previous generation, influences fear behaviour acquired from a short period of high risk as adults. We used fathead minnows as study subjects and simulated predation risk with repeated exposures to conspecific chemical alarm cues. The period of high risk experienced by adults induced typical fear behaviour (baseline freezing and neophobia), whereas the early-life low-risk period 1year prior caused only a reduction in baselineforaging. We found no evidence that the early-life risk significantly altered the fear acquired from the adult-risk period. However, in a second experiment, a low-risk period during the parental generation interacted with a high-risk period experienced by the adult offspring. The combination of both risk periods heightened baseline freezing despite parental risk having little effect independently. Hence, our study provides evidence that parental risk exposure can lead to an additive intergenerational effect on fear acquisition in minnows.

Predation risk mediates cognitive constraints following physical exertion in schoolmaster snapper

A large body of literature suggests that physically exhausted fish, including those that are released following fisheries interactions, experience behavioural and cognitive constraints and are at elevated risk of predation during homeostatic recovery. However, previous studies have focused on exhausted fish subsequently encountering predators, and not on fish that had been exposed to elevated predation risk prior to exhaustive exercise. Here, we exercised individual schoolmaster snapper (Lutjanus apodus) for 0, 1, or 4 min via hand chases following exposure to conspecific chemical alarm cues or seawater controls. The snapper were then introduced into one end of a rectangular arena supplied with mangrove prop roots as a refuge at the opposite end. Snapper exposed to the seawater control treatment demonstrated graded responses in mean times to move one body length and latency to enter the refuge, with unchased fish taking the least time and fish chased for 4 mins taking the longest. Amongst the snapper pre-exposed to alarm cues, the graded response did not occur and mean responses did not differ between chase treatments. Consistent with increased antipredator vigilance, alarm cue-exposed snapper were more likely to subsequently exit the refuge and to do so more times than fish exposed to seawater controls, independent of chase time. These observations suggest that perception of elevated predation risk may induce a conditional response offseting the behavioural and cognitive constraints associated with physical exhaustion through an unknown physiological mechanism to prioritize immediate survival-oriented behaviours over recovery.

Baseline activity and shoal type determine antipredator behaviors in bluegill from a southern Ontario lake

Shoaling is an evolved behavior in fishes that has several adaptive advantages, including allowing individuals to avoid predation through risk dilution. However, factors such as size disparity and the presence of heterospecifics may influence the behavior of individual fish within shoals following exposure to elevated predation risk. Using bluegill Lepomis macrochirus as a model species, we measured changes in area use, shoaling index, and movement of a focal individual in isolation, in single-species shoals with two conspecifics, or in mixed-species shoals with two congeneric pumpkinseed L. gibbosus. The experimental shoals were exposed to one of three chemical cues selected to present graded levels of risk: lakewater controls (lowest risk), Northern pike Esox lucius predator odor (kairomones; intermediate risk), or conspecific chemical alarm cues (highest risk). Within the individual bluegill, we found that the multivariate response of area use and post-stimulus activity (line crosses) of the focal fish was significantly influenced by pre-stimulus activity, but not by cue type or fish size. As univariate responses, post-stimulus activity varied positively with pre-stimulus activity. Post-stimulus activity was greater in single-species shoals compared to mixed-species shoals, and again varied positively with pre-stimulus activity. Contrary to predictions, bluegill did not demonstrate graded antipredator responses to the chemical cues. Our findings suggest that prey fish may alter their risk-aversive behaviors in response to chemical stimuli based on shoal composition and provide further insight into the role of intra-prey guild interactions in response to predators in co-occurring prey species. When faced with predation, individuals in groups may experience lower levels of risk than solitary individuals. Using bluegill as a model organism, we examined how antipredator behaviors, in response to chemical cues indicating different levels of risk, varied between focal individuals as singletons and in single- and mixed-species shoals. Contrary to our prediction of graded stepwise responses indicative of differing levels of risk posed by the cues, we found that individuals with greater baseline activity levels demonstrated weaker antipredator responses independent of cue type, while fish in single-species shoals had higher post-stimulus activity levels than fish in mixed-species shoals. Our results suggest that studies examining changes in antipredator and shoaling behaviors will benefit from including ecologically relevant scenarios involving sympatric prey guild members.

Do Gulf Toadfish Use Pulsatile Urea Excretion to Chemically Communicate Reproductive Status?

Gulf toadfish (Opsanus beta) are exceptionally capable of switching from excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses across the gill. Previous studies suggest that these urea pulses may be used for intraspecific chemical communication. To determine whether pulsatile urea excretion communicates reproductive status, toadfish were sexed using ultrasound and delivered conspecific-conditioned seawater (CC-SW) that previously housed a conspecific of the opposite sex, a conspecific chemical alarm cue (avoidance control), or a prey cue (attraction control). Swim behavior, attraction to or avoidance of the cues, and changes in the pattern of pulsatile urea excretion were monitored during and after delivery. Gulf toadfish did not spend more time in zones that were delivered CC-SW or prey cue. However, male toadfish spent significantly more time swimming after the delivery of female cues than control seawater (SW). In contrast, toadfish did not appear to have an immediate avoidance response to the conspecific alarm cue. Additionally, significantly more toadfish pulsed within 7 h of CC-SW and prey cue delivery compared to control SW, and pulse frequency was 1.6 times greater in response to CC-SW than control SW. These results, in combination with increased urea production and excretion the during breeding season, suggest that toadfish may use pulsatile urea excretion to communicate with conspecifics when exposed to chemosensory cues from the opposite sex.

Body condition, rather than size, predicts risk-taking and resource holding potential in hatchery reared juvenile lake sturgeon Acipenser fulvescens.

To test how body size might influence lake sturgeon Acipenser fulvescens anti-predator behaviour, asymmetrically body-size-matched individuals were exposed to conspecific chemical alarm cues with the presence or absence of food. Additionally, to test resource holding potential (RHP), hatchery-reared juvenile A. fulvescens were asymmetrically (c. 60% mass difference) and symmetrically (c. 3% mass difference) size matched in individual tanks. Results suggest that A. fulvescens of higher body condition, rather than body length or mass, may take greater risks when presented with foraging opportunities and realize a higher RHP. As hatcheries are likely to select individuals of higher body condition, more research is necessary to understand the role body condition may have on behavioural responses and ensuing fitness post-release.

Ventilation responses to predator odors and conspecific chemical alarm cues in the frillfin goby

The chemical detection of predation risk is direct when based on predator odors, or indirect when an injured conspecific or heterospecific signal it. Physiological adjustments may be necessary in parallel to defensive reactions to cope with an imminent risk. Here, we tested the effects of predator odors and conspecific chemical alarm cues in ventilation response (VR) of frillfin goby, Bathygobius soporator, because this response increases oxygen uptake for supporting behavioral tasks. No VR change was detected in response to odors of predators (catfish) that fed on conspecific, heterospecific fish (tilapia), or were deprived of food and to non-predator (tilapia) that fed chow (non-specific odor control) and odor eluent. The goby's VR, however, increased in response to conspecific alarm cues, but not to heterospecific cues or eluent. Clearly, the VR response in fish depends on the nature of the chemical cue. It is in line with ‘threat-sensitive hypothesis’ as a chemical cue from an injured prey might mean a foraging predator, whilst the mere presence of a predator odor might not. In addition, because VR can increase, decrease or remains unchanged in response to predation risk in other fish species (including other gobies), we reinforces the species-specific chracteristic of VR responses in fish, regarding the results obtained here for frillfin gobies.

Mianserin affects alarm reaction to conspecific chemical alarm cues in Nile tilapia.

In this study, I show that mianserin, a chemical with serotonin and adrenoceptor antagonist activities, increases fish vulnerability to a potential predator threat, when prey fish must deal with this threat based on conspecific chemical alarm cues. For that, I evaluated whether mianserin, diluted in the water, influences the behavioral responses of Nile tilapia (Oreochromis niloticus) to conspecific skin extract (chemical alarm cues). I found that, while mianserin did not abolished antipredator responses, this drug mitigates some components of this defensive reaction. Thus, a potential decrease in serotonin and adrenergic activities reduces the ability of dealing with predators when perceiving conspecific chemical alarm cues.

Interactive effects of reproductive assets and ambient predation risk on the threat-sensitive decisions of Trinidadian guppies.

Threat-sensitive behavioral trade-offs allow prey animals to balance the conflicting demands of successful predator detection and avoidance and a suite of fitness-related activities such as foraging, mating, and territorial defense. Here, we test the hypothesis that background predation level and reproductive status interact to determine the form and intensity of threat-sensitive behavioral decisions of wild-caught female Trinidadian guppies Poecilia reticulata. Gravid and nongravid guppies collected from high- and low-predation pressure populations were exposed to serial dilutions of conspecific chemical alarm cues. Our results demonstrate that there was `no effect of reproductive status on the response of females originating from a low-predation population, with both gravid and nongravid guppies exhibiting strong anti-predator responses to the lowest concentration of alarm cues tested. Increasing cue concentrations did not result in increases in response intensity. Conversely, we found a significant effect of reproductive status among guppies from a high-predation population. Nongravid females from the high-predation population exhibited a strong graded (proportional) response to increasing concentrations of alarm cue. Gravid females from the same high-predation population, however, shifted to a nongraded response. Together, these results demonstrate that accrued reproductive assets influence the threat-sensitive behavioral decisions of prey, but only under conditions of high-ambient predation risk.

Avoidance behavior in two sympatric planaria species: effects of conspecific and heterospecific chemical alarm cues

In many aquatic animals, predator avoidance can be stimulated by chemical cues, including those released by injured prey (alarm cues). Alarm cues of both conspecific and heterospecific origin have been identified within several fish taxa, where phylogenetic conservation of the cue-response complex is common. Turbellarian flatworms (planaria) are among the simplest animals known to respond to chemical cues released by injured conspecifics. We examined how two locally sympatric planaria species respond to conspecific and heterospecific chemical cues using macerated tissue suspensions. Brown (Girardia tigrina) and black (Dugesia dorotocephala) planaria both exhibited avoidance behavior when presented conspecific cues. Despite a significant twofold difference in body size (black > brown), stimulus prepared from a single (1×) individual of either species elicited avoidance. Increasing brown planaria cue concentration by macerating two individuals (2×) produced a significant increase in conspecific avoidance. Heterospecific stimuli produced asymmetric results. Black planaria avoided the brown planaria stimulus, but only in the higher concentration (2×) trials. Brown planaria did not consistently exhibit avoidance of the black planaria stimulus and some brown subjects approached and consumed black planarian tissues. Our results expand the demonstrated occurrence of alarm cues among planaria and suggest that avoidance behavior can be mediated by multiple environmental and intrinsic factors in freshwater Turbellaria.

Conspecific alarm cues affect interspecific aggression in cichlid fishes

Individuals have to respond simultaneously to different environmental factors often making trade-offs between conflicting demands necessary. Many freshwater ecosystems are resource-limited and both intra- and interspecific competitiveness is a common requirement to gain and defend resources necessary for reproduction. Although predation risk is an important selective force affecting behavioral decisions, little is known about the impact of predation risk on interspecific competition. Here, we investigate whether chemically mediated predation risk affects interspecific territorial aggression by the freshwater cichlid Pelvicachromis taeniatus. In our experiments, territorial P. taeniatus males were visually confronted with a territorial intruder: a heterospecific, sympatric cichlid (Benitochromis nigrodorsalis) which generally induced aggression in P. taeniatus. Predation risk for P. taeniatus was simulated by a concurrent release of conspecific chemical alarm cues. In control treatments, no chemical cues, dissolved heterospecific alarm cues, or aliquots of distilled water were provided during these aggressive encounters. The results show that interspecific aggression of territorial male P. taeniatus is significantly decreased under predation risk compared to the control treatments. This suggests that interspecific competition becomes less intense under concurrent predation risk. As this process could hinder competitive exclusion, predation risk may indirectly promote and stabilize biodiversity in natural ecosystems.

Active in the sac: damselfish embryos use innate recognition of odours to learn predation risk before hatching

Predation-induced mortality rates of aquatic species are much higher in larvae than in adults. Consequently, the ability of an organism to recognize relevant predators as early as possible could increase its chance of survival, especially in areas with high predator diversity. Heart rates of embryonic cinnamon clownfish, Amphiprion melanopus, were monitored to assess their reaction to damage-released conspecific alarm cues. These cues were then combined with a predator odour in a conditioning trial to establish whether the embryos were capable of learning a predatory threat. Results showed that A. melanopus embryos were not only able to detect and react to conspecific chemical alarm cues, but were also capable of using this information to learn about predation risk before they hatched. This recognition could lead to a number of antipredator behavioural adaptations, such as modifications of habitat choice at settlement, and could affect development and behaviour in postembryonic individuals, all of which may increase their chance of survival.

Conspecific alarm cue sensitivity by the estuarine calanoid copepod,Paracartia longipatella

Sensitivity to chemical cues associated with predation threat has been well observed in many freshwater zooplankters, yet few studies have highlighted such sensitivity in eury- and stenohaline metazoans. We aimed to assess sensitivity to conspecific chemical alarm cues in the estuarine copepod, Paracartia longipatella. Alarm cues associated with predation have been shown to have population level effects on certain zooplanktonic species. As such, we assessed the occurrence of such effects on population dynamics of P. longipatella over a 12 day period. Using experimental in situ mesocosms, we compared P. longipatella adult, copepodite and nauplii numbers between three treatments; one inoculated with conspecific alarm cues, one containing direct predation pressure (zooplanktivorous fish), and a control treatment containing no predation threat. Trends in population abundances were similar between the direct predation and alarm cue treatments for the six days of the experiment, decreasing in abundance. During the latter half of the study, however, P. longipatella abundances in the alarm cue treatment increased, while those in the presence of direct predation continued to decrease. In the treatment absent of any predation threat, P. longipatella abundances increased consistently over time for the duration of the study. We suggest that P. longipatella are indeed sensitive to conspecific alarm cues associated with predation threat. Furthermore, we propose that prolonged exposure to conspecific alarm cues in the absence of any real threat results in a reduction in sensitive to these cues.

Cortisol influences the antipredator behavior induced by chemical alarm cues in the Frillfin goby

We evaluated the effect of increased plasma cortisol levels on fish antipredator behavior induced by conspecific chemical alarm cues. The experimental model for the study was the Frillfin goby Bathygobius soporator. We first confirmed that the alarm substance induces typical defensive antipredator responses in Frillfin gobies and described their alarm substance cells (epidermal ‘club’ cells). Second, we confirmed that intraperitoneal cortisol implants increase plasma cortisol levels in this species. We then demonstrated that exogenous cortisol administration and subsequent exposure to an alarm substance decreased swimming activity to a greater extent than the activity prompted by either stimulus alone. In addition, cortisol did not abolish the sheltering response to the alarm chemical cue even though it decreased activity. As predators use prey movements to guide their first contact with the prey, a factor that decreases swimming activity clearly increases the probability of survival. Consequently, this observation indicates that cortisol helps improve the antipredator response in fish.

Fin‐flicking behaviour as a means of cryptic olfactory sampling under threat of predation

Risk of predation is a ubiquitous component of behavioural decision-making (Lima & Dill 1990). For small littoral fishes, chemical cues released from predators and/or injured conspecifics, comprise important sources of information about the presence and nature of predation risk (see Kelley 2008; Ferrari et al. 2010 for recent reviews). Behavioural responses to these chemical cues include area avoidance, increased shoal cohesion, reduction in activity, movement out of the water column, use of shelter and finflicking (Lawrence & Smith 1989; Ferrari et al. 2010). Collectively, these behavioural responses reduce the probability of predation (Mathis & Smith 1993). Fin-flicking is a behaviour in which fish quickly sweep their pectoral fins anteriorly, matched with concomitant counterbalancing forward thrusts of the anal and caudal fin, resulting in no net movement. The function of fin-flicking has not received much attention in the context of antipredator behaviour. Fin-flicking by glowlight tetras (Hemigrammus erythrozonus, Durbin 1909) in response to conspecific chemical alarm cues induced greater shoal cohesion in conspecifics and also deterred attack behaviour by a potential predator, the Jack Dempsey cichlid (Rocio octofasciata, Regan 1903; Brown et al. 1999). Be that as it may, the precursor function of fin-flicking signals may be more utilitarian in nature and only later became co-opted into a visual signalling system. This brief communication presents a test of the hypothesis that fin-flicking by alarmed fish serves the selfish benefit of allowing individuals to sample chemical cues in the surrounding waters whilst remaining motionless to avoid attracting attention from predators. Receptors that detect chemical alarm cues and predator odours are arranged in rosettes in the nares, which are blind pouches in the rostrum (Doving & Lastein 2009). Advection (mass water movement) of water to the nares is generated by ram ventilation as fish move through the water column. When risk is detected, fish are faced with a trade-off because the physical swimming movements needed to assess predation risk are conspicuous to predators. Fin-flicking behaviour may be a solution to this trade-off if it allows individuals to discreetly create microcurrents of water past the nares resulting in continuous sampling of chemical information of the surrounding water without the accompanying movements that would compromise crypsis. Java moss (Taxiphyllum barbieri) was chopped into 2-mm pieces to create small neutrally buoyant particles that would allow us to visualise water currents generated by fin-flicking. In one recording session, an adult fathead minnow (Pimephales promelas, Rafinesque 1820) was placed in a shallow white plastic trough that afforded good visual contrast with the moss fragments. In a second recording session, an adult fathead minnow was placed into a narrow aquarium with a small amount of clay to visualise water currents. Skin extract was prepared by killing an adult fathead minnow by cervical dislocation and then lightly scoring its flanks with a clean razor blade. One minnow was killed for each session of filming. The minnow carcasses were then soaked in 100 ml of deionised water for 10 min. The test dose of alarm cue was 10 ml of minnow skin extract administered to the side of the container to induce fin-flicking behaviour. All procedures were approved by the Minnesota State University Moorhead Institutional Animal Care

Learning temporal patterns of risk in a predator-diverse environment.

Predation plays a major role in shaping prey behaviour. Temporal patterns of predation risk have been shown to drive daily activity and foraging patterns in prey. Yet the ability to respond to temporal patterns of predation risk in environments inhabited by highly diverse predator communities, such as rainforests and coral reefs, has received surprisingly little attention. In this study, we investigated whether juvenile marine fish, Pomacentrus moluccensis (lemon damselfish), have the ability to learn to adjust the intensity of their antipredator response to match the daily temporal patterns of predation risk they experience. Groups of lemon damselfish were exposed to one of two predictable temporal risk patterns for six days. “Morning risk” treatment prey were exposed to the odour of Cephalopholis cyanostigma (rockcod) paired with conspecific chemical alarm cues (simulating a rockcod present and feeding) during the morning, and rockcod odour only in the evening (simulating a rockcod present but not feeding). “Evening risk” treatment prey had the two stimuli presented to them in the opposite order. When tested individually for their response to rockcod odour alone, lemon damselfish from the morning risk treatment responded with a greater antipredator response intensity in the morning than in the evening. In contrast, those lemon damselfish previously exposed to the evening risk treatment subsequently responded with a greater antipredator response when tested in the evening. The results of this experiment demonstrate that P. moluccensis have the ability to learn temporal patterns of predation risk and can adjust their foraging patterns to match the threat posed by predators at a given time of day. Our results provide the first experimental demonstration of a mechanism by which prey in a complex, multi-predator environment can learn and respond to daily patterns of predation risk.