Yellowtail damselfish Chrysiptera parasema can associate predation risk with the acoustic call of a heterospecific damselfish following pairing with conspecific alarm cues.

The ability to detect and respond to the presence of predation risk is under intense selection, especially for small-bodied fishes. Damselfishes (Pomacentridae) use auditory vocalizations during inter- and intrasexual interactions, but it is not known if they can use vocalizations in the context of predator-prey interactions. Here, we test if yellowtail damselfish, Chrysiptera parasema, can learn to associate the territorial vocalization of heterospecific humbug damselfish Dascyllus aruanus with predation risk. In conditioning trials yellowtail damselfish were presented with the territorial call of humbug damselfish while either blank water (control treatment) or chemical alarm cue derived from damaged skin of conspecific yellowtail damselfish was introduced. In conditioning trials, fish exposed to alarm cue exhibited increased activity and spent more time in the water column relative to fish that received the control treatment. After a single conditioning trial, conditioned fish were exposed again to the territorial call of humbug damselfish. Fish conditioned with the call + alarm cue showed increased activity and spent more time in the water column relative to fish that had been conditioned with the control treatment. These data indicate associative learning of an auditory stimulus with predation risk in a species that regularly uses auditory signalling in other contexts. Recordings of conditioning and test trials failed to detect any acoustic calls produced by test fish in response to the perception of predation risk. Thus, although yellowtail damselfish can associate risk with auditory stimuli, we found no evidence that they produce an alarm call.

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.

Zebrafish embryos hatch early in response to chemical and mechanical indicators of predation risk, resulting in underdeveloped swimming ability of hatchling larvae.

Plasticity in hatching time allows embryos to maximize fitness by balancing the benefits and costs of remaining bound within the chorion against the benefits and costs of emerging as a free-swimming larva. Here, in the first experiment, we exposed zebrafish (Danio rerio) embryos to either chemical cues from crushed embryos (simulating egg predation) or to blank water control. Embryos exposed to alarm cues hatched sooner, and had shorter body lengths and underdeveloped fins, relative to larvae from the water treatment. Burst swimming speed was significantly slower for larvae that hatched from the alarm cue treatment than for larvae from the water treatment. In a second 2×2 experiment, we exposed zebrafish embryos to either chemical alarm cues from conspecific embryos, mechanical disturbance (magnetic stir bar) to simulate a predator probing the substrate for developing embryos, both chemical and mechanical indicators of risk, or neither (control). We found similar effects in terms of earlier time to hatch at an earlier stage of development and poorer swimming performance of hatchling larvae. In the second experiment, these effects occurred in response to mechanical disturbance with or without the presence of chemical alarm cues. Alarm cues alone produced no effects in the second experiment. Taken together, these data indicate that zebrafish embryos demonstrate a facultative trade-off between risk of predation acting on two stages of their life history.

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.

Association of predation risk with a heterospecific vocalization by an anabantoid fish.

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 the 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.

Tone deaf: Association of an auditory stimulus with predation risk by zebrafish Danio rerio does not generalize to another auditory stimulus.

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.

Inside the Undergraduate Immunology Classroom: Current Practices that Provide a Framework for Curriculum Consensus.

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.

Epidermal Club Cells in Fishes: A Case for Ecoimmunological Analysis.

Epidermal club cells (ECCs), along with mucus cells, are present in the skin of many fishes, particularly in the well-studied Ostariophysan family Cyprinidae. Most ECC-associated literature has focused on the potential role of ECCs as a component of chemical alarm cues released passively when a predator damages the skin of its prey, alerting nearby prey to the presence of an active predator. Because this warning system is maintained by receiver-side selection (senders are eaten), there is want of a mechanism to confer fitness benefits to the individual that invests in ECCs to explain their evolutionary origin and maintenance in this speciose group of fishes. In an attempt to understand the fitness benefits that accrue from investment in ECCs, we reviewed the phylogenetic distribution of ECCs and their histochemical properties. ECCs are found in various forms in all teleost superorders and in the chondrostei inferring either early or multiple independent origins over evolutionary time. We noted that ECCs respond to several environmental stressors/immunomodulators including parasites and pathogens, are suppressed by immunomodulators such as testosterone and cortisol, and their density covaries with food ration, demonstrating a dynamic metabolic cost to maintaining these cells. ECC density varies widely among and within fish populations, suggesting that ECCs may be a convenient tool with which to assay ecoimmunological tradeoffs between immune stress and foraging activity, reproductive state, and predator-prey interactions. Here, we review the case for ECC immune function, immune functions in fishes generally, and encourage future work describing the precise role of ECCs in the immune system and life history evolution in fishes.

Factors Contributing to Sex Differences in Mice Inhaling Aspergillus fumigatus.

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.

Evidence for incipient alarm signalling in fish.

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".

Olfactorily-mediated cortisol response to chemical alarm cues in zebrafish Danio rerio.

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.

Emotional response of undergraduates to cadaver dissection.

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.

Bold, Sedentary Fathead Minnows Have More Parasites.

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.

Shoaling as an antiparasite defence in minnows (Pimephales promelas) exposed to trematode cercariae.

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 group-living. 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.

High intensity and prevalence of two species of trematode metacercariae in the fathead minnow (Pimephales promelas) with no compromise of minnow anti-predator competence.

Opportunity for parasites to manipulate host behavioral phenotype may be influenced by several factors, including the host ecology and the presence of cohabiting parasites in the same host. Metacercariae of Ornithodiplostomum ptychocheilus and "black spot" Crassiphiala bulboglossa have similar life cycles. Each parasite uses a littoral snail as a first intermediate host, fathead minnows as a second intermediate host, and a piscivorous bird as a final host. Metacercariae of black spot encyst in the dermal and epidermal tissues, while metacercariae of O. ptychocheilus encyst on the brain over a region that coordinates optomotor responses. Because of site differences within the host, we predicted that O. ptychocheilus metacercariae might manipulate the behavioral phenotype of minnows to facilitate transmission to the final host, but metacercariae of black spot would not. In our study population, prevalence was 100% for O. ptychocheilus , with an overall median intensity of 105 metacercariae per minnow. Prevalence of black spot was 60%, with a median abundance and intensity of 12 and 20 metacercariae per minnow for the overall sample and for infected fish, respectively. Minnows accumulated both parasites over time, producing significant correlations between intensity and minnow body length and between intensities of the 2 parasites. Minnows infected with black spot had on average twice as many O. ptychocheilus metacercariae as similar-sized minnows without any black spot cercariae. We found no correlation between body condition of minnows and intensity for either parasite. We measured 2 aspects of anti-predator competence to test for effects linked to parasite intensity. We found no correlation between intensity of either species of parasite and latency to behavioral response to attack from a mechanical model heron, nor was there any effect of parasite intensity on a measure of shoaling affinity. The absence of any detectable effect of metacercariae on anti-predator competence in minnows may reflect selection against parasite pathology from predation by non-hosts of the parasites and overwinter mortality due to low dissolved oxygen.

Epidermal 'alarm substance' cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation.

Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.

Field verification of predator attraction to minnow alarm substance.

Fishes such as minnows in the superorder Ostariophysi possess specialized alarm substance cells (ASC) that contain an alarm cue. Alarm substance can only be released by damage to the epidermis; thus, the release of alarm substance is a reliable indicator of predation risk. When nearby minnows detect the cue, they adopt a range of antipredator behaviors that reduce their probability of predation. Predator-predator interactions afford prey an opportunity to escape and, thus, a fitness benefit that maintains alarm substance calls over evolutionary time. Here, we present data from a simple field experiment verifying that nearby predators are attracted to minnow alarm substance because it signals an opportunity to pirate a meal. Fishing lures were baited with sponge blocks scented with either (1) water (control for sponge odor and appearance), (2) skin extract from non-ostariophysan convict cichlids (superorder Acanthopterygii, Archocentrus "Cichlasoma" nigrofasciatus) to control for general injury-released cues from fish, or (3) skin extract from fathead minnows (superorder Ostariophysi, Pimephales promelas). Predator strike frequency on each sponge type was 1, 1, and 7 for water, cichlid, and minnow cues, respectively. These data provide the first field test using fish predators of the predator-attraction hypothesis for the evolution of Ostariophysan alarm substance cells.