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Many fishes learn to recognize correlates of predation risk by pairing novel stimuli with injury-released chemical cues released from damaged epidermal tissues. Here, zebrafish were conditioned to associate predation risk with a three-note auditory stimulus C5 (523.25 Hz) + E5 (659.25 Hz) + G5 (783.99 Hz), then tested for responses to either only one tone (C), two of the components (C + G) or the full three-note chord (C + E + G). Zebrafish conditioned with alarm cues and C + E + G responded significantly more intensely to the full C + E + G stimulus or to partial representation of the original mix (C + G) than they did to the single element (C) of the original C + E + G conditioning stimulus. The lack of a response to the single component alone may be a failure to recognize the stimulus or an interpretation that the partial component is an indicator of low risk.

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.

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.

Honey gouramis (Trichogaster chuna) received chemical alarm cues derived from conspecific epidermal tissue and, simultaneously, the vocalization produced by a heterospecific gourami species, the sparkling pygmy gourami (Trichopsis pumila). Control trials paired water with the vocalization. In trials that received alarm cues, honey gouramis significantly increased activity relative to control trials that received water, suggesting an attempt to flee and search for refuge. When the recording of the vocalization was later replayed to test fish without any additional chemical cue, fish that had previously experienced alarm cue froze while those that had received water with the vocalization did not change their behaviour. These data indicate that honey gouramis recognize and respond to chemical alarm cues, making this report the second anabantoid species to be recorded with this response. Second, these data indicate that honey gouramis can associate risk of predation with a novel auditory stimulus, including vocalizations from other species. These data suggest the potential for vocalizations to evolve into alarm signals in this group of fishes. This article is protected by copyright. All rights reserved.

Wisenden et al. (2020) reported differential behavioral responses by male Red-winged Blackbirds (Agelaius phoeniceus) to models bearing a red-epaulet visual signal with and without reflectance in the ultraviolet (UV) portion of the spectrum. O'Daniels et al. (2021) wrote a response, claiming that some of our key terminology was incorrect and that our methodology was potentially confounded, calling into question the veracity of the results reported in our study. Herein, we explain (1) our use of the term “control” to be the treatment that lacks the test variable of interest; (2) the labels UV+and UV–refer to treatments that did, and did not, reflect in the UV; (3) that sunscreen likely did not contain methyl anthranilate, and even if it did, the amounts of sunscreen used were so small that it could not possibly explain the behavioral effects we observed over the distances between models and territorial males tested in the field; and (4) visual modeling would not enhance the interpretation or impact of our study. We do agree with O'Daniels et al. (2021) that additional field research should be done to better understand visual ecology and the evolution of avian plumage signals.

Although immunological research has become increasingly important in recent decades for understanding infectious and immune-mediated diseases, immunological pedagogy at the undergraduate level has lagged behind in reports of evidence-based scholarship. To address the need for a renewed emphasis on immunology education and to describe the current status of undergraduate education in immunology, an online survey of instructors with experience in teaching immunology was conducted. The survey investigated the effects of instructors' level of teaching experience, target student population, and course components on the emphasis given to certain immunology subtopics in their courses. Instructor teaching experience and current role in teaching influenced the proportion of time allotted to lab techniques, clinical topics, and evolutionary aspects, but type of institution (undergraduate and graduate degree-granting institutions) did not affect course content or emphasis on subtopics. Topics that received the greatest emphasis were the adaptive immune system, the innate immune system, host-pathogen interactions, and molecular mechanisms. Vaccines, hypersensitivity, autoimmunity, and essential immunology techniques were ranked slightly lower, while topics such as evolution, metabolism and antibody purification received the least emphasis. Inclusion of a lab component increased time given to lab-related and clinical topics but did not affect the perceived importance of various scientific competencies. These data describe current curricular practices of instructors who have experience teaching immunology and inform curricular priorities and course design frameworks for undergraduate immunology education.

Aspergillus fumigatus is a respiratory fungal pathogen and an allergen, commonly detected in flooded indoor environments and agricultural settings. Previous studies in Balb/c mice showed that repeated inhalation of live and dry A. fumigatus spores, without any adjuvant, elevated allergic immune response and airway remodeling. Sex-specific differences can influence host-pathogen interactions and allergic-asthma related outcomes. However, the effect of host sex on immune response, in the context of A. fumigatus exposure, remains unknown. In this study, we quantified the multivariate and univariate immune response of C57BL/6J mice to live, dry airborne A. fumigatus spores. Our results corroborate previous results in Balb/c mice that repeated inhalation of live A. fumigatus spores is sufficient to induce mucus production and inflammation by day 3 post last challenge, and antibody titers and collagen production by day 28 post-challenge. Principal Component Analysis (PCA) showed that females exhibited significantly higher levels of immune components than males did. Taken together, our data indicate that host-sex is an important factor in shaping the immune response against A. fumigatus, and must be considered when modeling disease in animals, in designing diagnostics and therapeutics for A.fumigatus-associated diseases or while drafting evidence-based guidelines for safe mold levels.

Predator recognition by small fishes can be acquired when chemical alarm cues released from damaged skin (by a predator attack) are paired with a novel stimulus, such as the appearance or odor of a predator. Once learned, fish can extend recognition of risk by generalizing to associate risk with additional stimuli that are similar to the conditioned novel stimulus. Here, we trained zebrafish to associate a novel auditory stimulus with predation risk, and then tested to see if they generalize risk to all sound stimuli or whether the conditioned response is limited to the sound frequency of the conditioning stimulus. We found that zebrafish Danio rerio readily associated risk of predation with Tone 1 (285 Hz), as evidenced by reduction in activity, increased time spent near the substratum and increased shelter use, but fish conditioned to fear Tone 1 completely ignored presentation of a second tone of 762 Hz. These data suggest that generalization does not occur as easily for auditory cues as they do for olfactory and visual cues, perhaps due to differences in the properties of sensory biology or the cognitive mechanisms that process information in different sensory modalities.

The most effective way to learn human anatomy is through cadaver dissection. Historically, cadaver dissection has been the provenance of professional schools. Increasingly, cadaver-based courses in human anatomy are shifting to the undergraduate level, which creates both problems and opportunities because of differences between undergraduate and graduate student populations. Anxiety associated with dissecting cadavers can create a barrier to learning, and ultimately, entry into the health and medical sciences for some demographic subpopulations of undergraduates. We surveyed 76 students in 2007 and 51 students in 2009 at four times in the semester to investigate the timing and sociodemographic predictors of anxiety over cadaver dissection. We followed this with a second survey of 44 students in 2014 to test the effect of humanization of cadaver donors (providing information about donor occupation and cause of death) to reduce student anxiety. Students experienced anxiety upon first exposure to cadaver dissection. Female students experienced greater anxiety than male students upon first exposure to cadavers but this effect was short-lived. Self-identified non-white, non-Christian students experienced sustained anxiety throughout the semester, likely because cadaver stress compounded social and financial stressors unique to international students. Humanization was effective in reducing anxiety in non-white, non-Christian students but had the unexpected effect of increasing anxiety in female students. We recommend that humanizing information be offered to students who seek it out, but not forced upon students for whom the information would only add to their stress. Clin. Anat. 31:224-230, 2018. © 2017 Wiley Periodicals, Inc.

Chemical characterization of damage-released alarm cues in ostariophysan fishes has lagged far behind the study of the ecological role that these cues play in behavioral decision-making of prey fishes. Chondroitin sulfate has been identified as a putative component of alarm cue based on two laboratory studies of zebrafish, Danio rerio, and the northern studfish, Fundulus catenatus. The fathead minnow, Pimephales promelas, is a model organism in the study of chemically mediated predator–prey interactions, in part because they can be studied in the laboratory and under field conditions. Here, we conducted a field experiment on wild populations of fathead minnows, to test for area avoidance of chondroitin sulfate relative to conspecific skin extract (containing alarm cues = positive control) or water (negative control). We repeated the experiment in two small lakes in central Minnesota using minnow traps containing blocks of sponge with one of the three test cues. We found that fathead minnows avoided traps chemically labeled with conspecific alarm cue more than control traps labeled with water, and that the number of minnows caught in traps labeled with chondroitin sulfate was intermediate between alarm cue traps and water traps. These data are consistent with laboratory findings that chondroitin sulfate is a component of alarm cue, but that other species-specific compounds are needed for a full behavioral response.

Summary 1 Laboratory-based research provides great control over individual experimental replicates, but lacks the ecological realism of field work. Therefore, results from lab-based work may not reflect natural processes. Field-based studies have the advantage of ecological realism but often lack control over environmental variables and have low rates of within-experiment replication and as a result can have low statistical power to detect effects. 2 Here, we present a method for creating a standardized system for experimental replicates in the field using solar-powered water pumps to run a modified zebrafish aquatic habitat system with recirculating water in situ. In our pilot study we used this apparatus to test the effects of naturally-occurring parasites on the behavior of individual fathead minnows, but this experimental set-up has potential for broad application to field-based research in aquatic ecology. 3 We constructed a simple frame from conventional 2” x 4” lumber to support rows of 1-L tanks in the littoral zone of a small lake. Lake water is pumped to an overhead manifold from a PVC pipe drilled with one hole over each tank. A short length of airline tubing directs the water flow into each individual tank. Water entering each tank passes through the manufacturer's grate at the rear of each tank and returns to the lake. We stocked one fish per tank, therefore the lack of water flow during the night did not cause duress or mortality. 4 This system allowed control over individual test subjects while housing them in semi-natural conditions with a steady supply of lake water at a field site far removed from any source of electricity. This article is protected by copyright. All rights reserved.

In Focus: Bairos-Novak, K.R., Ferrari, M.C.O., & Chivers, D.P. (2019). A novel alarm signal in aquatic prey: Familiar minnows coordinate group defences against predators through chemical disturbance cues. Journal of Animal Ecology, 88, 1281-1290, https://doi.org/10.1111/1365-2656.12986. Chemicals released during predation have long been studied as cues to nearby prey that predators are active in the vicinity. Until now, these chemicals have been labelled as cues because there was no compelling evidence for the necessary components of a communication system, namely (a) voluntary control of release of information, (b) capacity for graded responses and (c) the presence of specialized structures for the production and release of the signal. New findings by Bairos-Novak, Ferrari, and Chivers (2019) show that fathead minnows alter the potency of disturbance "cues" when in the presence of other fathead minnows compared to when they are alone and produce either more or different disturbance "cues" when in the presence of familiar conspecifics compared to when they are in the presence of unfamiliar conspecifics. The behavioural response to these cues is shoaling, which would confer fitness benefits to the sender, thereby qualifying as a signal rather than a cue. This is a significant advancement in the field of chemical ecology of aquatic organisms because disturbance "cues" by fathead minnows bear two of the three hallmarks of an incipient disturbance "signal".

Parasites that rely on trophic transmission can manipulate the behavior of an intermediate host to compromise the host's antipredator competence and increase the probability of reaching the next host. Selection for parasite manipulation is diminished when there is significant risk of host death to causes other than consumption by a suitable definitive host for the parasite. Consequently, behavioral manipulation by parasites can be expected to be subtle. Ornithodiplostomum ptychocheilus (Op) is a trematode parasite that has a bird-snail-fish host life cycle. Fathead minnows are a common intermediate host of Op, where metacercariae encyst in the minnow brain. In this study, we report a link between metacercarial intensity and behavior in fathead minnows. In the field, we found that roaming distance by free-living minnows over 24 h was negatively correlated with parasite intensity. In the laboratory, we found that boldness in an open field test was positively correlated with parasite intensity. These parasite-induced behavioral changes may render infected minnows more susceptible to predators, which would serve to facilitate trophic transmission of parasites to the bird host.

Parental care in fishes is a three-way interaction among brood predators, parental brood defence, and the escaping ability of the developing young. Convict cichlids are Neotropical freshwater fish with prolonged biparental brood defence of their eggs and free-swimming larvae. In a previous study, developmental timing of changes in larval swimming performance was correlated with larval skeletal ossification and biparental brood defence in convict cichlids in the Rio Cabuyo, a stream in Costa Rica (Wisenden et al. 2015). Here, we repeat this study on a population of convict cichlids from Laguna de Xiloa, a volcanic crater lake in Nicaragua. We found that fish from Laguna de Xiloa also showed correlations among swimming performance and skeletal ossification of the larvae, and brood defence by the parents. However, in Laguna de Xiloa the developmental timing of these events was delayed relative to the Rio Cabuyo population. The population difference between Costa Rica and Nicaragua could be an effect of genetic divergence or an artefact of phenotypic plasticity between lab-reared fish (Costa Rica) versus wild-caught fish (Nicaragua) for ossification scores. To resolve this question we repeated study using lab-reared fish of brood stock from Laguna de Xiloa. Comparing among the lab-reared Costa Rican fish, and lab-reared and field-collected Nicaraguan fish, we found that the timing of ossification was significantly delayed for both Nicaraguan samples relative to the Costa Rican samples. These shifts likely reflect population differences in selection on anti-predator competence of the young and, consequently, parental brood defence. These data indicate that larval ontogenetic development, anti-predator performance and parental care co-evolve with site-specific ecological differences.

Male Red-winged Blackbirds (Agelaius phoeniceus) have red epaulets that are used as badges for intrasexual competition for territory acquisition and defense. Red epaulets reflect very litt...

Chemical cues released as a by-product of predation mediate antipredator behaviour, but little is known about the physiological responses to olfactory detection of predation risk. In this study, zebrafish Danio rerio were exposed to either chemical alarm cues from conspecifics, or water (control). Compared with water controls, D. rerio exposed to alarm cues responded behaviourally with antipredator behaviours such as erratic dashing and an increase in time spent near the bottom of the test aquarium. Danio rerio were sacrificed 5 min after exposure to test cues (alarm cues or water). Enzyme-linked immunosorbent assay (ELISA) revealed whole-body levels of cortisol that were significantly higher for fish exposed to alarm cues (mean ± SE, 11.9 ± 3.4 ng g-1 ) than control fish (1.5 ± 0.7 ng g-1 ). These data provide a benchmark for future studies of the proximate mechanisms of olfactorily mediated antipredator responses, modelling effects on aquatic life in a changing climate and, as a model organism, Danio rerio can further our understanding of anxiety in humans.

In a field study of Leon Springs pupfish Cyprinodon bovinus, two questions about female promiscuity were investigated. First, were females selective in the males with whom they spawned or were they unselective, spawning randomly among males? Second, how promiscuous were the females, i.e. with how many males did they spawn? If simply spawning with many males maximized a female's reproductive success, then females might be expected to spawn randomly with as many males as possible. Alternatively, if females were selective but engaged in multiple mating, they would limit their spawning to preferred males. In the only wild population of this endangered fish, breeding males defend closely associated territories in the shallow margins of a single desert pool. No territories were observed elsewhere in the pool. Therefore, all territorial males were present simultaneously and females could survey all of them, depositing any number of eggs with one, a few or many males. Rather than spawning randomly, females surveyed many males first, visited relatively few males and ultimately spawned with a small fraction of those available males. With increasing numbers of spawns, however, females increased the number of different mates with whom they spawned. Thus, females showed a bet-hedging tactic of having a narrow mate preference while also laying eggs in the territories of other males, possibly to reduce egg predation and to avoid inbreeding.

Mobile young under parental care have a high potential for intermixing with other broods, which potentially increases the costs to the foster parents. Here, we examined for the first time the genetic composition of wild-caught broods of the convict cichlid (Amatitlania siquia), a socially monogamous biparental fish, for evidence of brood mixing and adoption. Our microsatellite genotyping data revealed that 79% of broods contained adopted young. Moreover, 25% of broods contained adopted sibsets likely arising from extra-pair matings, a phenomenon hitherto not documented for this species. Overall, adopted foreign fry and host fry in mixed broods were generally different in body length, as would be expected if they have different parents. However, fry from possible extra-pair matings were similar in body length to host fry, suggesting that they are of similar age. Our results are important because they reveal a very high prevalence and degree of brood mixing, and indicate that social monogamy does not necessarily lead to genetic monogamy in the convict cichlid in nature. These findings raise questions about potential brood-mixing mechanisms and the reproductive ecology (especially opportunities for polygamy in nature) of this important model species in the study of animal behaviour.

Convict cichlids are Neotropical freshwater fish with biparental brood defense of their free-swimming young. In this study, we correlate ontogenetic changes in swimming performance measured by maximum velocity and burst speeds with skeletal ossification of larvae and consider how these data provide insights into the proximate mechanisms for the evolution of parental care. In lab tests, swimming velocity and acceleration of the young increased nonlinearly with body length with a rapid improvement at 7 mm in standard length (SL). The timing of abrupt improvement in swimming velocity and acceleration coincided with the timing of skeletal ossification from cartilage to bone. Radii of defended broods in the field reflected the interaction of parental defense competence and offspring antipredator competence. In the Rio Cabuyo, Costa Rica, brood radii increased steadily until the young were 6.45 mm SL, and then plateaued. Taken together, these data indicate interdependent and co-evolved traits that encompass larval ontogenetic development, antipredator performance, habitat-specific predation regime, and parental care.

Individual zebrafish Danio rerio were assayed for exploratory tendency in a serial open field test before and after being maintained on one of four diet treatments that differed in ration and in predictability of food delivery. Danio rerio became more exploratory after being maintained on a diet with a predictable delivery schedule. There was no effect of ration. Thus, exploratory behaviour is inducible by environmental influences independent of genetic predisposition or social interactions. These results have implications for management of correlated behavioural syndromes of exploratory and boldness of animals reared in captivity for later release into wild populations.

Prey fishes use chemical cues in their predator's diet to cue the context and timing of antipredator behaviors that reduce the risk of predation. They also use dietary cues to learn to recognize and later detect the presence of a predator. Because dietary chemical information decreases hunting success of predators there should be selection on predators to mask or obliterate these cues through biochemical processes in their digestive tract. In this study, we compared response intensity of predator-naive fathead minnows to the signature odors of two novel predators that had been fed either a minnow diet or a non-minnow diet. Unprocessed minnow mash and dechlorinated blank water were used as positive and negative controls, respectively. One predator was from a relatively ancestral fish group, the order esociformes, while the other predator was from a relatively derived group, the order perciformes. Naive minnows were able to detect the presence of minnows in the diet of the ancestral and derived predators wi...

Predation exerts tremendous selection pressure on all organisms. In this study, we exposed embryos of convict cichlids (Amatitlania siquia) twice daily to one of the following: (1) chemical alarm cues of damaged conspecifics + odour of a novel predator (Polypterus endlicheri), (2) chemical alarm cues of damaged conspecifics + water or (3) blank water. No chemical cues were presented after the eggs hatched. When the larvae were 9 days old (mean total length = 5.7 mm), they were exposed to either predator odour or water. Those larvae that had been conditioned as embryos on alarm cues + predator odour showed a significant reduction in activity (i.e. anti-predator behavioural response) to predator odour relative to the other treatments. This is the first demonstration of acquired predator recognition by fish embryos.

Summary 1. Individuals that live in groups benefit from increased foraging success and decreased predation. Protection from some types of parasites may provide an additional benefit of groupliving. For fish, the extent to which shoaling can reduce an individual’s risk of exposure to the infective stages of parasites is unknown. 2. We tested for antiparasite benefits of shoaling in fathead minnows exposed to larvae (cercariae) of two of their most common species of trematode, Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum. As developing stages (metacercariae) of these trematodes cause reductions in minnow activity, growth and survival, natural selection should favour the evolution of cercariae-avoidance behaviours. 3. We evaluated shoal dimensions in groups of minnows exposed to O. ptychocheilus and to other chemical/physical stimuli within aquaria. To compare risk of exposure in shoaling vs. non-shoaling fish, we confined groups of minnows into mesh cages in outdoor mesocosms, exposed them to cercariae, then compared mean worm numbers in grouped vs. solitary fish. Lastly, we tested whether fish located within the centre of an artificial shoal reduced their risk of cercariae exposure compared with those along peripheral edges. 4. Minnows distinguished infective cercariae from other potential aquatic threats and responded with activity that reduced the 2-dimensional area of their shoals 15-fold compared with water-only controls. Fish confined within artificial shoals had 3-fold fewer worms than single fish and minnows located within the centre of artificial shoals had significantly fewer worms than those without peripheral minnows. 5. These results show that shoaling reduces a minnows’ risk of exposure to cercariae, either directly via detection of cercariae in the water column followed by behavioural avoidance or indirectly via behaviour-mediated differences in exposure between shoaling vs. non-shoaling fish.

Hatcheries are effective at producing large numbers of fish for augmenting fisheries or conserving endangered populations, but the fish they produce are often predator-naive, resulting in high levels of predation mortality when the fish are first released into natural water bodies. Fish normally acquire recognition of novel stimuli as indicators of danger when injury-released chemical cues from conspecifics (a known indicator of an actively foraging predator) are presented simultaneously with a novel stimulus (e.g., predator odor, image). Thus, fish in wild populations quickly learn the sight and smell of their predators. Past research has demonstrated that predator-naive, hatchery-reared fish can be trained to recognize predators and that fish trained by this method have a significantly greater probability of surviving an encounter with a predator. To implement predator training in fishery management, predator recognition training must be feasible on a large scale in a way that does not place an...

Correlated behavioural traits (behavioural syndromes) influence ecological interactions and, in turn, how natural selection shapes the evolution of behaviour. A major axis of intraspecific behavioural variation is the exploratory-boldness behavioural syndrome. Here, we describe how we sorted wild-caught zebrafish (Danio rerio) for increasing exploratory tendency using a serial open field maze. We then transferred test fish to a predator-inspection apparatus. Highly exploratory individuals spent more time near a novel predator than non-exploratory individuals, showing behavioural consistency indicative of an exploratory-boldness behavioural syndrome. We then bred the zebrafish to create F1 offspring from four crosses: (1) exploratory × exploratory, (2) non-exploratory × non-exploratory, (3) exploratory females × non-exploratory males and (4) non-exploratory females × exploratory males. F1 offspring were tested in a miniature serial open-field maze similar to the one used to sort the adults. Offspring of exploratory females were always highly exploratory regardless of the behavioural traits of the father suggesting that maternal effects may influence expression of exploratory-boldness behavioural syndrome in zebrafish.

The interaction between predator and prey is an evolutionary arms race, for which early detection by either party is often the key to success. In aquatic ecosystems, olfaction is an essential source of information for many prey and predators and a number of cues have been shown to play a key role in trait-mediated indirect interactions in aquatic communities. Here, we review the nature and role of predator kairomones, chemical alarm cues, disturbance cues, and diet cues on the behaviour, morphology, life history, and survival of aquatic prey, focusing primarily on the discoveries from the last decade. Many advances in the field have been accomplished: testing the survival value of those chemicals, providing field validation of laboratory results, understanding the extent to which chemically mediated learning may benefit the prey, understanding the role of these chemicals in mediating morphological and life-history adaptations, and most importantly, the selection pressures leading to the evolution of chemical alarm cues. Although considerable advances have been made, several key questions remain, the most urgent of which is to understand the chemistry behind these interactions.

In aquatic environments, chemical cues released during a predator attack reliably inform prey about the presence of predation risk. Prey with information about predation risk are more successful in surviving encounters with predators than are unwary prey. To remain prepared for attack, prey should continue to monitor the status of predation risk, presenting a behavioural trade-off for prey: increased distance from areas labelled with alarm cues reduces exposure to predation risk but also reduces access to information about predation risk. In two laboratory experiments we used the presence and absence of water flow in a laboratory fluvarium to test alarm response and subsequent risk-sensitive information gathering by zebrafish (Danio rerio). In response to chemical alarm cues, fish significantly reduced activity and increased use of shelters. In the absence of flow, fish sought out the shelter nearest the cue source. In the presence of flow, fish preferred to seek shelter downstream, but not upstream, of the cue source. This allowed fish to gather information about predation risk from a relatively safe distance. In a field experiment on natural populations of stream fishes, fish avoided areas where chemical alarm cues were released (versus blank water control) but primarily because they avoided the region immediately upstream of the cue source. Fish use of the area immediately downstream of cue release did not decrease. Taken together, these laboratory and field data are consistent with a trade-off between risk avoidance and information gathering.

Parental investment in unrelated offspring is potentially maladaptive but may be promoted by natural selection if the presence of foreign young enhances the survival of the parents' own young. We experimentally augmented broods of free-ranging convict cichlids (Cichlasoma nigrofasciatum) to test whether survival of the adopting parents' young (fry) increases, in relation to that of control broods, after the addition of smaller foreign fry, and whether such an increase can be attributed to the effect of brood dilution acting alone or to a combination of brood dilution and the effect of differential predation on adopted young. Total fry survival did not differ between experimental (E) broods and control (C) broods, but E broods had significantly more large (host) fry after 5 days and 10 days than C broods did. In E broods, small (foreign) fry suffered higher rates of predation than large fry, indicating differential predation. In E broods starting at 7.0 and 7.5 mm standard length (SL), observed fry mortalities did not differ significantly from mortalities expected from the effect of brood dilution. However, E broods starting at 8.0 mm SL had significantly lower mortalities than expected, indicating that parents that adopt smaller foreign fry can increase the survival of their own fry by the combined effects of brood dilution and differential predation. Within E broods, growth of smaller foreign fry was significantly slower than that of larger host fry, suggesting that intra-brood agonistic behaviour affects access to food for smaller fry. Therefore, increased predation and reduced growth are two negative effects that act on fry of donor parents.

In aquatic environments, chemical cues serve as an important source of information for the detection of predation risk. Here, we investigate the response of convict cichlids, Cichlasoma nigrofasciatum, to injury-released chemical cues. We exposed pairs of juvenile convict cichlids first to dechlorinated tap water (control), then later to one of two test stimuli: 1. chemical cues from injured convict cichlids; or 2. chemical cues from injured mosquito fish, Gambusia affinis. Gambusia are allopatric and phylogenetically unrelated to convict cichlids. Gambusia skin was used to control for a general response to injured fish. In response to conspecific cues, convict cichlids significantly increased time spent near the bottom of test aquaria and time under a shelter object. In response to Gambusia skin, convict cichlids tended to increase time spent near the tank bottom but did not increase use of the shelter object. There was a trade-off between antipredator and agonistic behaviours. In response to convict cichlid cues, there was a significant reduction in the frequency of approaches and bites. Gambusia skin extract had no significant effect on aggressive behaviour. These data suggest a species-specific antipredator response to conspecific alarm pheromones in a New World cichlid fish and demonstrate a trade-off between predator avoidance and intraspecific aggression. Further, the presence of an alarm response in this model species sets the stage for the use of chemical cues as a research tool to manipulate predation risk in studies of the interaction between predation risk and reproductive behaviour.

In this study we tested the effects of predation risk on reproductive behaviour of Iowa darters (Etheostoma exile). We recorded the frequency of courtship behaviour (head shaking) and spawning acts for 30 min before and after removing an opaque barrier to expose groups of darters to (1) an empty tank (the before-pike treatment), then (2) a tank containing a pike (the pike treatment), and finally (3) an empty tank (the after-pike treatment). In response to the pike, darters performed significantly less reproductive behaviour than before exposure to the pike. Furthermore, darters performed proportionally less reproductive behaviour in the area in the immediate vicinity of the pike where risk of predation was highest. However, darters continued to occupy the risky area while exposed to the pike. Removal of the barrier in the absence of the pike (the before- and after-pike treatments) had no effect on the overall level of reproductive behaviour, the proportion of reproductive behaviour performed in the immediate vicinity of the pike tank (the risky area), or use of the risky area.

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

Epidermal club cells of fishes in the superorder ostariophysi have puzzled evolutionary biologists because they were historically linked to chemical alarm signalling and relied on group selectionist explanations. Alternative hypoth- eses to explain the existence of these cells include the possibility of an anti-pathogenic or anti-parasitic function. If this is so, individual fish should invest in increased numbers of club cells after exposure to parasites, and club cells should contain components that reduce the infectivity of skin-penetrating larvae. Infectivity of cercariae of the trematode Ornithodiplostomum sp. was significantly reduced when exposed to the skin extract of fathead minnows (an ostariophysan), but also to skin extract of mollies (a non-ostariophysan that lacks club cells), respectively, compared to controls. Moreover, club cell density was not affected by exposure to cercariae. Taken together, these results are inconsistent with an anti-parasite function for these cells and instead suggest a generic role in response to injury. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 884-890. ADDITIONAL KEYWORDS: alarm substance - anti-predation - Ornithodiplostomum - Ostariophysi - infectivity - Pimephales.

A behavioral syndrome is a suite of behaviors correlated across multiple social contexts. In this study, boldness in the face of predation risk was assessed twice in fish across two different sensory modalities in both the field and lab to ascertain the biological relevance and complexity of this attribute. Individual fathead minnows were captured from a natural field population using traps that either contained chemical alarm cues (conspecific skin extract) or control (well water) and their responses to the presence of predator behind a glass partition assessed in the laboratory. Although fewer fish were captured in alarm cue-labeled traps, these bold fish performed longer predator inspections than shy fish captured in control traps. Thus, a shy/bold behavioral syndrome was expressed consistently across field and lab settings in response to both chemical and visual indicators of danger. Shy and bold individuals did not differ in sex, body length, secondary sexual characteristics, or parasite load but were of more robust physical condition.

Summary Predation risk is detected by the presence of cues released passively during a predation event. Refinement of risk assessment could occur if prey have the ability to assess cue age. Here, we test for antipredator behavioural responses to chemical alarm cues of varying ages. Fathead minnows, Pimephales promelas, give an antipredator response to alarm cues derived from conspecifc skin extract that has been aged at 18 ◦ C for 0 (fresh) and 3 h, but not after 6 h. Alarm cues from crushed conspecific Gammarus lacustris (Crustacea: Amphipoda), showed a similar chemical longevity. A field test of minnow alarm cues produced broadly similar results for conspecific fathead minnows and heterospecific responses by northern redbelly dace, Phoxinus eos. Close agreement among all three data sets suggests either a common duration of predation risk or a common molecular basis of chemical cues across aquatic taxa. When we heated skin extract to denature and remove half of the constituent proteins, we inactivated biological activity of alarm cue suggesting that protein is required for skin extract to function as an alarm cue in minnows. Protein degradation may be a means of assessing age and, therefore, ecological relevance of chemical information in behavioural decision-making.

We studied parental care behaviour in a field population of convict cichlids, Amatitlania siquia, a fish with biparental care of its free-swimming young (fry). We simulated catastrophic brood loss by removing all of the young from 26 broods of various ages. We recorded parental defence behaviour for 5 min preremoval and three consecutive 5 min postremoval intervals. We then returned all of the young and recorded 5 min of parenteoffspring reunion behaviour. Following brood loss, both parents defended the empty territory and searched for missing young. Females spent more time in the empty territory and performed more attacks in defence of the empty territory than did males. Females searched and defended a significantly larger area than males did. During the 15 min fry-absent period, males increasingly spent more time away, whereas females maintained a constant level of investment in searching the brood area. Four of 26 males (15%) engaged in courtship with a new female within 15 min of brood loss. Attacks to repel potential predators from the brood area declined in frequency over the 15 min fry-absent period in both sexes. Standard length (age) of young was positively correlated with parental investment for females but not males. Number of young was positively correlated with parental investment for males but not for females. We conclude that post-brood-loss behaviours are search and recovery tactics that contribute to parental inclusive fitness in the event of incomplete brood predation.

Fathead minnows Pimephales promelas exposed to cercariae of the trematode Ornithodiplostomum sp. incurred a significant mass loss 17 days after exposure to 20 or 120 cercariae. Parasite-naive P. promelas showed no evidence of innate recognition or avoidance of cercariae. After a single exposure to cercariae, however, fish responded to chemical and visual cues of dead (thawed) cercariae with a reduction in activity. Encounter rate with cercariae, and hence infection rate, increased with fish activity. The data indicated that experienced P. promelas associated parasitism risk with novel chemical and visual cues that later triggered avoidance behaviour. Parallels and interactions between antiparasite behaviour and antipredator behaviour open new avenues for behavioural ecological research in risk-sensitive decision-making.

In this study, we tested the tendency of young cichlids to orient towards their parents using chemical cues. Convict cichlids have biparental defence of their free-swimming young. If young become separated from their family they must rejoin their family within minutes to have any chance of survival. Here, we used a dichotomous Y-maze to test if displaced convict cichlid young can use chemical cues to orient to their family. First, we showed that young preferentially orient toward water taken from their home tank versus blank water taken from a tank that contained no fish. Second, we showed that young prefer home tank water to water from a tank containing another family of convict cichlids. In a third experiment, we placed young convict cichlids in a small aquarium inserted within a larger aquarium. The large aquarium contained either their parents or no fish. The small insert aquarium contained blank water from a tank that had never contained fish. After 20 min in the insert tank, young convict cichlids were placed in the Y-maze and given a choice between either their home tank water or water from the insert tank. Convict cichlids that did not see their parents during the 20-min conditioning period oriented strongly toward chemical cues of their home tank water. Young that saw their parents during the 20-min conditioning period oriented strongly toward the chemical cues of the insert tank. These data indicate that young convict cichlids use visual cues to learn and reset the smell of “home” in less than 20 min.

Chemical cues released from injured fish skin during a predator attack provide reliable information about the presence of predation risk. Here, I report estimates of the area avoided by littoral fishes after experimental release of chemical alarm cues in two small lakes in northern Minnesota. Minnow traps were labeled chemically with either water (control) or skin extract (chemical alarm cue) made from 2 cm2 of cyprinid skin (redbelly dace in experiment 1, fathead minnows in experiment 2). Traps labeled with water were placed 1, 2, or 8 m from traps labeled with alarm cue. After 2 h, water-traps that were either 1 or 2 m distant from an alarm-trap caught significantly fewer fish than water-traps 8 m distant from alarm-traps. Conspecific and heterospecific skin extract produced similar area avoidance by fathead minnows. Redbelly dace showed a larger active space in response to conspecific than heterospecific alarm cues. Brook stickleback showed reduced catches within 2 m of skin extract of fathead minnows. Overall, the radius of active space was between 2 and 8 m under lake conditions with average subsurface currents of 0.82 cm/s. These data are the first field estimates of active space of ostariophysan chemical alarm cues.

When a predators attack prey, damaged prey tissue releases chemical information that reliably indicates an actively foraging predator. Prey use these semiochemicals to cue anti-predator behaviour and reduce their probability of predation. Here, we test central mudminnows, Umbra limi (Kirtland 1840), for anti-predator behavioural responses to chemical cues in conspecific skin extract. In a field experiment, traps scented with mudminnow skin extract (alarm cue) caught fewer mudminnows than traps scented with water (control). Under controlled laboratory conditions, mudminnows showed a significant reduction in activity and movement to the bottom in response to alarm cues relative to water controls. Reduced activity and increased time on the bottom of the tank are both known components of an anti-predator response. Thus, based on field and lab data, mudminnows exhibited anti-predator behavioural responses to chemical alarm cues released by damaged epidermal tissue. Histological preparations of epidermal tissue did not reveal the presence of specialised “alarm substance” cells for the production of chemical alarm cues. This is the first report of an alarm reaction in an esociform, an order with a long evolutionary history of piscivory.