Anti‐predator response of naïve and experienced common bully to chemical alarm cues

Histological analysis of the skin of common bully Gobiomorphus cotidianus , a New Zealand native eleotrid fish, revealed the presence of club cells in the epidermis. Epidermal club cells are frequently associated with the production of alarm substance (Schreckstoff). The behavioural responses of perch‐naïve and perch‐experienced common bullies to either conspecific skin extract or chemical cues from an introduced predator, perch Perca fluviatilis , were then examined. Both perch‐naïve and perch‐experienced common bullies exhibited a behavioural response when exposed to conspecific skin extract, indicating the probable presence of an alarm substance. In contrast, only perch‐experienced common bullies recognized and exhibited a subsequent behavioural response to the odour of perch. This study is the first to document the presence of epidermal club cells and a behavioural response to a conspecific chemical alarm signal for fishes in the Eleotridae. The results indicate that common bully can learn to recognize perch odour as a threat, and that this ability may be a result of previous predator labelling involving a conspecific alarm substance.     

Feeding and Growth Response of Roach, Rutilus rutilus, to Alarm Substance

Feeding and growth responses of roach from three size classes to alarm substance (Schreckstoff) were quantified in laboratory experiments. Larger fish (60.0–80.0 mm in length) reacted stronger to treatment than two smaller sized groups (35.0–45.0 and 46.0–55.0 mm) lowering feeding rate by 80 and 40 and 50%, respectively. The reduction in feeding rate of larger fish caused decrease in growth rate in length and weight, while the lowered consumption of smaller fish caused only reduction in growth rate in weight. Condition factor of exposed to alarm substance small sized roach was lower than that of the control individuals and roach from other two size classes, both, treated and untreated. The difference in growth response to a danger of predation has its roots probably in different metabolism and growth rates of small and large fish. Small fish have higher metabolic rate and less lipid reserves than larger ones, therefore they are probably forced to feed to be able to grow. Also, small sized roach is more vulnerable to predation than large sized fish, thus growing fast seem to be crucial for survival in a risky environment. Study shows that small roach trade off their safety against food, feeding in risky environment to sustain fast growth. This ability of fast outgrowing of a dangerous, vulnerable to predators, size increases survival of juveniles in dangerous environment.     

Effects of alarm substance on schooling in the common shiner (Notropis cornutus,Cyprinidae)

Synopsis The behavioral responses of common shiners (Notropis cornutus) to Schreckstoff (alarm pheromone produced in the epidermis) were quantified in laboratory experiments. Schreckstoff increased cohesion and polarization, and decreased the variability in overall school dimensions. There was also a positive correlation between size of fish and their distance to the center of the school. Furthermore, the tendency to seek cover increased in the presence of Schreckstoff. The greater organization in school structure appears to be an adaptive response to aquatic predators, whereas increased cover seeking may be an adaptive response to aerial predators.     

Socializing makes thick-skinned individuals: on the density of epidermal alarm substance cells in cyprinid fish, the crucian carp (Carassius carassius)

In cyprinid fish, density of epidermal club cells (i.e. alarm substance cells) has been found to vary between lakes with different predator fauna. Because predators can be labelled with chemical cues from prey, we questioned if club cell density could be controlled indirectly by predators releasing prey cues. In particular, we suspected a possible feedback mechanism between chemical alarm signals and their cellular source. We raised crucian carp singly and in groups of four. For both rearing types, fish were exposed to skin extracts of either conspecifics or brown trout (without club cells), and provided either low or high food rations. Independent of rearing type, condition factor and club cell density increased with food ration size, but no change was found in club cell density following exposure to conspecific alarm signals. However, the density of club cells was found significantly higher for fish raised in groups than for fish raised alone. We conclude that an increased condition factor results in more club cells, but crucian carp may also possess an awareness of conspecific presence, given by higher club cell densities when raised in groups. This increase in club cell density may be induced by unknown chemical factors released by conspecifics.     

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.     

Fluoreszenzmikroskopischer Nachweis des Schreckstoffes in den Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.) (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Die Schreckstoffzellen der Elritze,Phoxinus phoxinus (L.), zeigen nach Gefriertrocknung der Haut eine Eigenfluoreszenz. Diese wird bei 360–380 nm maximal angeregt; das Emissionsmaximum liegt bei ca. 515 nm. Die Fluoreszenz einer Schreckstoffzelle wird mit der Fluoreszenz des isolierten Schreckstoffes mikrospektralphotofluorimetrisch verglichen: die Emissions-maxima liegen nur 10 nm voneinander entfernt. Die Kurven relativer Fluoreszenzintensität verlaufen weitgehend gleichartig. Dies zeigt, daß der Schreckstoff tatsächlich den Schreckstoffzellen entstammt.

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Fluorescence microscopical demonstration of the alarm substance in the alarm substance cells of the European minnow,Phoxinus phoxinus (L.) (cyprinidae, ostariophysi, pisces)

Summary The alarm substance cells of the European minnow,Phoxinus phoxinus (L.), are autofluorescent after freeze-drying of the skin (Fig. 1). Their autofluorescence is maximally excited at 360–380 nm; the maximum of emission lies at about 515 nm. The fluorescence of an alarm substance cell is compared with the autofluorescence of the isolated alarm substance by means of a microspectro-photofluorometer. The maxima of emission are only about 10 nm apart. The curves of relative fluorescence intensity are almost identical (Fig. 3). These results show that the alarm substance actually comes from the alarm substance cells.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Predator recognition and defence strategies in crucian carp, Carassius carassius

Crucian carp from populations that lack piscivores are extremely vulnerable to predation. However, in the presence of piscivores these fish develop an inducible morphological defence, a deep body. This switch from a vulnerable, shallow-bodied morph to a morphologically defended morph makes this species very suitable for investigations of anti-predator strategies, and trade-offs between morphological and behavioural defences. To address these questions, we performed eight different experiments. We found that crucian carp exhibited fright responses to chemical cues from unfamiliar predators (northern pike, perch) when these were fed prey that contained alarm substance (for northern pike: crucian carp, roach; for perch: crucian carp). Cues from small pike that were fed prey that lacked alarm substance (swordtails) caused no significant fright response whereas cues from larger pike with the same diet did. Perch on a chironomid diet elicited weaker but significant fright responses. Starved predators caused as strong fright reactions as recently fed ones did, whereas no response was exhibited towards nonpredatory fish (roach, crucian carp). Crucian carp were able to detect the presence of pike after cues had been diluted to an equivalent of 21 000 l, and larger predators elicited stronger fright responses. Prior experience of predators decreased fright responses. In particular, individuals from populations that coexisted with northern pike responded less to chemical cues from northern pike than individuals without prior experience did. Thus, crucian carp may use both alarm-substance related and predator-related cues to identify predators. Further, they were able to discriminate between large and small predators. Finally, individuals from populations that coexist with predators exhibit less pronounced fright responses. These fish have an induced morphological defence, a deep body, which most likely decreases the need for strong antipredator behaviour.     

The Influence of Food Competition and Prédation Risk on Size-assortative Shoaling in Juvenile Chub (Leuciscus cephalus)

Size-assortative shoaling is a widespread phenomenon potentially linked to competition for resources among shoal members and anti-predator benefits gained from living in shoals. This paper investigates the influence of both factors on size-assortative shoaling in chub (Leuciscus cephalus). In a first experiment food competition was studied by manipulating the proportions of small and large chub in shoals of 20 fish. Large fish were generally more competitive than small ones. However, the competitive effect of large fish was strongly dependent on their number in the shoal. This has important consequences for the theory of competition because it makes it difficult to predict stable combinations of different phenotypes when foragers divide between food patches. Despite the strong effects of food competition no significant trend for size-assortativeness was observed in free-swimming shoals when undisturbed. This changed after the shoal had been frightened by introducing Schreckstoff (an alarm substance) to the test tank. Large and small fish still remained in the same shoal but showed a strong preference to be close to neighbours of their own size class. Large fish were observed mainly in the centre of the shoal and small fish were found more in the periphery. It is unclear whether these position differences are due to aggressive behaviour of large fish towards small fish which excludes them from the potentially safe centre of the shoal. These results indicate that prédation plays a more important role than food competition for size-assortative shoaling in chub.     

Challenging fear: chemical alarm signals are not causing morphology changes in crucian carp (<Emphasis Type="Italic">Carassius carassius</Emphasis>)

Crucian carp develops a deep body in the presence of chemical cues from predators, which makes the fish less vulnerable to gape-limited predators. The active components originate in conspecifics eaten by predators, and are found in the filtrate of homogenised conspecific skin. Chemical alarm signals, causing fright reactions, have been the suspected inducers of such morphological changes. We improved the extraction procedure of alarm signals by collecting the supernatant after centrifugation of skin homogenates. This removes the minute particles that normally make a filtered sample get turbid. Supernatants were subsequently diluted and frozen into ice-cubes. Presence of alarm signals was confirmed by presenting thawed ice-cubes to crucian carp in behaviour tests at start of laboratory growth experiments. Frozen extracts were added further on three times a week. Altogether, we tested potential body-depth-promoting properties of alarm signals twice in the laboratory and once in the field. Each experiment lasted for a minimum of 50 days. Despite growth of crucian carp in all experiments, no morphology changes were obtained. Accordingly, we conclude that the classical alarm signals that are releasing instant fright reactions are not inducing morphological changes in this species. The chemical signals inducing a body-depth increase are suspected to be present in the particles removed during centrifugation (i.e., in the precipitate). Tissue particles may be metabolized by bacteria in the intestine of predators, resulting in water-soluble cues. Such latent chemical signals have been found in other aquatic organisms, but hitherto not reported in fishes.     

Untersuchungen zur Isolierung und Identifizierung des Schreckstoffes aus der Haut der Elritze,Phoxinus phoxinus (L.) (Cyprinidae,Ostariophysi, Pisces)

Zusammenfassung Es wurde versucht, den Schreckstoff aus der Haut der Elritze,Phoxinus phoxinus (L.) (Cyprinidae), zu isolieren und zu identifizieren. Hierfür wurden die einzelnen Fraktionen im Verhaltensexperiment an Schwärmen des Malabarbärblings,Danio malabaricus (Jerdon) auf ihren Schreckstoffgehalt geprüft. Nach der dünnschichtchromatographischen Auftrennung der reinsten Fraktionen löste nur das Eluat von zwei stark fluoreszierenden Flecken die Schreckreaktion aus. Die Ergebnisse von Vergleichsuntersuchungen an zwei weiteren Species, nämlich dem Malabarbärbling (Cyprinidae) und dem SalmlerAlestes longipinnis (Günther) (Characidae), stimmten mit den an der Elritze gewonnenen Befunden völlig überein. Beim Schreckstoff handelt es sich sehr wahrscheinlich um ein Pterin. Isoxanthopterin wurde als Vergleichssubstanz getestet und löste ebenfalls die Schreckreaktion aus.

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On the isolation and identification of the alarm substance from the skin of the European minnow,Phoxinus phoxinus (L.) (cyprinidae, ostariophysi, pisces)

Summary The alarm substance from the skin of the European minnow,Phoxinus phoxinus (L.) (Cyprinidae), was isolated and identified. The amounts of alarm substance in various fractions of the skin were tested by means of behavioural experiments on schools of the giant danio,Danio malabaricus (Jerdon). From all thinlayer chromatograms tested, only two strongly fluorescent spots elicited the fright reaction. The results of comparative investigations on two other species, the giant danio (Cyprinidae) and the long-finned African tetraAlestes longipinnis (Günther) (Characidae), were in complete agreement with the results onPhoxinus. The results indicate that the alarm substance very probably is a pterin. Comparative tests with isoxanthopterin also elicited the fright reaction.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Parasites,info-disruption,and the ecology of fear

There is growing interest in the ecological consequences of fear, as evidenced by the numerous studies on the nonconsumptive, trait-mediated effects of predators. Parasitism, however, has yet to be fully integrated into research on the ecology of fear, despite it having direct negative and often lethal effects on hosts and being the most common life history strategy on the planet. This might at least be partly due to the traditional, but untested, assumption that anti-parasite responses are weak relative to anti-predator responses. To test this hypothesis, we quantified the activity and location responses of Bufo americanus tadpoles to one of six chemical cues: water; cercariae of Echinostoma trivolvis, a trematode which infects and can kill amphibians; a snail releasing E. trivolvis cercariae; an uninfected snail; food; or conspecific alarm chemicals signaling predation. There is also literature encouraging research on the context dependency and pollution-induced disruption of fear responses. Consequently, before quantifying responses to the chemical cues, half of the B. americanus were exposed to the herbicide atrazine (201 μg/l for 4 days), a reported inhibitor of fear responses in fish. Tadpoles were attracted to food, were indifferent to an uninfected snail, avoided alarm chemicals, and exhibited avoidance and elevated activity in response to a snail shedding cercariae and cercariae alone. Atrazine had no detectable effects on B. americanus’ responses to the tested cues despite the use of a higher concentration and longer exposure duration than has been repeatedly shown to inhibit chemical cue detection in fish. The magnitude of anti-parasite and anti-predator responses were qualitatively similar, suggesting that the fear of disease and its ecological consequences could be comparable to that of predation. Consequently, we call for a greater integration of parasites into research on the ecology of fear and trait-mediated indirect effects.     

Role of olfaction and vision cues in feeding behavior and alarm reaction in the catfish pintado, Pseudoplatystoma corruscans

Experiments were performed to investigate senses that are essential for mediating fright reaction and food behavior in Pseudoplatystoma corruscans, pintado. The dilemma “to feed or to flee” was also analyzed in fishes with intact and sectioned olfactory tracts, stimulated by alarm substance extracts and food. Fishes were arranged into five groups: fish with intact lateral olfactory tracts (LOT), fish with intact medial olfactory tract (MOT), fish with tracts totally sectioned (TOTAL, both LOT and MOT), sham operated, and nonoperated fish. The five groups were submitted to either alarm substance extract and food stimulus or to distilled water (control) and food stimulus. Fish reacted to food independently of which tract (LOT, MOT or TOTAL) was sectioned; vision seems necessary and elemental to detect and deflagrate food response. Latency of the responses to each reaction was different between groups. None of the fish with sectioned tracts reacted to alarm substance extract, while sham- and nonoperated fish showed the typical alarm behavior response, leading to the conclusion that olfaction is essential for mediating alarm response. These results indicate that others sense systems (e.g., vision) are sufficient to trigger and elicit feeding behavior and that olfaction is not necessary to fully maintain food detection to qualitative and quantitative extent. However, olfactory tract integrity seems to be required for mediation of alarm reaction in P. corruscans.     

Effects of steroid hormones and other substances on alarm substance cells and mucous cells in the epidermis of the European minnow, Phoxinus phoxinus (L.), and other Ostariophysi (Pisces)

The alarm substance cells and mucous cells of various species of Ostariophysi do not react uniformly to treatment with androgen and other steroid hormones. This indicates that the mechanism controlling formation of alarm substance is not uniform in the Ostariophysi and three exist at least two such mechanisms, one of which is operated by androgens and perhaps other steroid hormones of the gonads. Steroid hormones from the adrenal cortex proved ineffective. In the European minnow, anti-androgen (androcur®, cyproteroneacetate) had an efrect opposite to that of androgen, while androgen treatment upon this species led to an increase in the size of mucous cells and a reduction in the alarm substance cells. The epidermis of androgen-treated European minnows contained neither alarm substance cells nor alarm substance, and skin extracts of such fish proved ineffective in behaviour experiments upon fish schools. The effect of further hormone treatments are discussed and the reactions of the European minnow are compared to those of other species.     

Alarm cue induces an antipredator morphological defense in juvenile Nicaragua cichlids Hypsophrys nicaraguensis

Olfactory cues that indicate predation risk elicit a number of defensive behaviors in fishes, but whether they are sufficient to also induce morphological defenses has receivedlittle attention. Cichlids are characterized by a high level of morphological plasticity during development, and the few species that have been tested do exhibit defensive behaviors when exposed to alarm cues released from the damaged skin of conspecifics. We utilized young juvenile Nicaragua cichlids Hypsophrys nicaraguensis to test if the perception of predation risk from alarm cue (conspecific skin extract) alone induces an increased relative body depth which is a defense against gape-limited predators. After two weeks of exposure, siblings that were exposed to conspecific alarm cue increased their relative body depth nearly double the amount of those exposed to distilled water (control) and zebrafish Danio rerio alarm cue. We repeated our measurements over the last two weeks (12 and 14) of cue exposure when the fish were late-stage juveniles to test if the rate of increase was sustained; there were no differences in final dimensions between the three treatments. Our results show that 1) the Nicaragua cichlid has an innate response to conspecific alarm cue which is not a generalized response to an injured fish, and 2) this innate recognition ultimately results in developing a deeper body at a stage of the life history where predation risk is high [Current Zoology 56 (1): 36-42, 2010].     

The ‘club’ cell and behavioural and physiological responses to chemical alarm cues in the Nile tilapia

The alarm response to skin extract has been well documented in fish. In response to skin extract, there is a decline in both locomotion activity and aggressive interactions. Our observation herein of these responses in the cichlid Nile tilapia, Oreochromis niloticus, confirmed the existence of the alarm response in this species. However, so far there has been a paucity of information on the autonomic correlates of this response. In this study, the ventilatory change in response to the chemical alarm cue was evaluated. This parameter was measured 4 min before and 4 min after exposure to 1 mL of either conspecific skin extract or distilled water (extract vehicle). Skin extract induced an increase in the ventilation rate, which suggested an anticipatory adjustment to potentially harmful stimuli. The chemical cue (alarm substance) also interfered with the prioritisation of responses to different environmental stimuli (stimuli filtering); this was suggested by the observation that the Nile tilapia declined to fight after exposure to a cue that indicates a risk of predation. Furthermore, histological analysis of the Nile tilapia skin revealed the presence of putative alarm substance-producing (club) cells.     

Reaction to Alarm Substance in Cave Populations of Astyanax fasciatus (Characidae,Pisces)1

The effect of species-specific skin-extract containing alarm substance has been studied in two epigean and three hypogean populations of the Mexican-tetra Astyanax fasciatus. In all populations tested the feeding rate at the water surface decreased significantly after introducing the alarm substance and the fish avoided this surface area. Additionally epigean fish and some individuals from the Chica cave showed zigzag movements, rapid swimming and hiding. The fish did not respond to a skinextract of a species without alarm substance.     

Who dares, learns: chemical inspection behaviour and acquired predator recognition in a characin fish

Individuals that dare approach predators (predator inspection behaviour) may benefit by acquiring information regarding the potential threat of predation. Although information acquisition based on visual cues has been demonstrated for fish, it is unknown whether fish will inspect predators on the basis of chemical cues or whether such inspection behaviour results in information acquisition. Here, we first ascertained whether predator inspection behaviour can be mediated by chemical cues from predators by exposing groups of predator-naive glowlight tetras (Hemigrammus erythrozonus) to the chemical cues of a potential fish predator (convict cichlid Cichlasoma nigrofasciatum) that had been fed either tetras (which possess an alarm pheromone) or swordtails (Xiphophorus helleri, which lack Ostariophysan alarm pheromones). Tetras showed a significant increase in antipredator behaviour when exposed to the tetra-diet cue, but not when exposed to the swordtail-diet cue. Chemically mediated predator inspection behaviour was also affected. Both the latency to inspect and the minimum approach distance to the predator significantly increased, and the mean number of inspectors per predator inspection visit significantly decreased when tetras were exposed to the tetra-diet versus the swordtail-diet chemical cues. We then examined a potential benefit associated with chemically mediated predator inspection behaviour. Only tetras that were initially exposed to the tetra-diet cue and that had inspected the predator acquired the visual recognition of a convict cichlid as a predation threat. Our results thus demonstrate that (1) predator inspection behaviour in the glowlight tetra can be initiated by chemical cues, (2) chemically mediated inspection behaviour is affected by the presence of alarm pheromone, and (3) inspectors benefit by acquiring the recognition of novel predators. Copyright 1999 The Association for the Study of Animal Behaviour.     

Inducible defences in Daphnia depend on latent alarm signals from conspecific prey activated in predators

Some water fleas (Daphnia spp.) undergo phenotypic changes when exposed to chemical signals from predators. The chemical signals have been assumed to be of predator origin (i.e. kairomones), since juices of crushed Daphnia have been found ineffective. We speculated that latent alarm signals could be present in Daphnia, to be activated in predators following ingestion. Accordingly, fish predators were fed earthworms for 10 weeks to remove Daphnia remains from their gastro-intestinal tracts. Following another 6 days of earthworm feeding, water conditioned by fish induced no morphological changes in D. galeata. When fish were alternatively fed Daphnia for 6 days, changes were induced with fish-conditioned water. Extracts made from intestines of earthworm-fed fish, homogenized with earthworms, gave no morphological changes, but intestines of the same origin homogenized with Daphnia did. Similar results were found when earthworms and Daphnia were homogenized with fish liver. Freshly frozen extracts of homogenized Daphnia gave no detectable changes at first instar stage in test animals, whereas extracts of Daphnia that had been kept at room temperature did induce such changes. Our results suggest that Daphnia respond to latent conspecific alarm signals (i.e. 'dormant' pheromones) that are activated by intestinal or bacterial enzymes in predators or in the water.     

Alarm reaction in the crucian carp is mediated by the medial bundle of the medial olfactory tract

Experiments were performed to determine which bundles of the olfactory tracts were essential for mediating alarm reaction in crucian carp (Carassius carassius L.). The fish were maintained in physiological saline after surgery to preserve the remaining tracts and postoperative inspections revealed the functionality of the intact tracts. Operations on the tracts were performed symmetrically on both sides. Sham-operated and non-operated fish showed the typical alarm behaviour of fast swimming to the bottom, dashing movements and aggregation when exposed to skin extract which contain alarm substance. Fish with only the medial bundle of the medial olfactory tract intact also displayed the alarm behaviour upon exposure; however, these fish did not react to the amino acid, L-alanine with either feeding response or alarm reaction. Crucian carp which had the medial bundle of the medial olfactory tract cut, leaving both the lateral bundle of the medial olfactory tract and the lateral olfactory tract intact, did not display any alarm reaction to skin extract; however, these fish reacted to exposure to L-alanine with feeding behaviour. There were statistically significant differences between the behaviour scores for the fish subject to different treatments. The present study demonstrates that the medial bundle of the medial olfactory tract appears to be both necessary and sufficient for mediation of the alarm reaction. The results also show that the sensory neurons which respond to alarm substance terminate and make synaptic connections with the secondary neurons that make up the medial bundle of the medial olfactory tract; thereby demonstrating the specificity of the spatial aspect of olfactory processing. The results are discussed with respect to the spatial aspect of organization within the olfactory system, the pattern of generalization across orders of fish, and the functional implications of the spatial arrangement of information transmission between the peripheral olfactory organ and the brain.     

Acquired Recognition of Predator Odour in the European Minnow (Phoxinus phoxinus)

Naive European minnows (Phoxinus phoxinus) do not show a fright reaction when they first encounter the odour of a natural predator (the pike: Esox lucius) or the odour of a non-piscivorous exotic (tilapia: Tilapia mariae). A conditioned fright response to both these odours will however develop if minnows experience them in a potentially dangerous situation, for example, in conjunction with Schreckstoff, the ostariophysian alarm pheromone. Although minnows respond to both odours the reaction to the tilapia odour is reduced. This suggests that a constraint on learning is involved. Olfactory recognition is particularly valuable for detecting predators that hunt in conditions where visibility is poor.