Background predation risk induces neophobia in zebrafish

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Crane, Ahmad Ghobeishavi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4477880/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Oct, 2024 Read the published version in Animal Cognition → Version 1 posted 9 You are reading this latest preprint version Abstract Prey face a major challenge in balancing predator avoidance with other essential activities. In environments with high risk, prey may exhibit neophobia (fear of novelty) due to the increased likelihood of novel stimuli being dangerous. The zebrafish, Danio rerio , is an established model organism for many scientific studies. Although spatial and object neophobia in zebrafish have received previous attention, little is known about the role of background risk in inducing neophobia in zebrafish. Here, we present two experiments using zebrafish to explore whether background predation risk can induce fear in a novel environment and when exposed to a novel odour. Over five days, we repeatedly exposed zebrafish to either high background risk in the form of chemical alarm cues (i.e., injured conspecific cues that indicate a predator attack) or a low-risk water control stimulus. When tested in a novel spatial environment, zebrafish exposed to high predation risk displayed fear responses (reduced activity and bottom time) compared to their low-risk counterparts. Moreover, high-risk individuals exhibited fear responses toward a novel odour, unlike low-risk individuals. These results reveal that short-term repeated exposures to high risk can induce neophobia in zebrafish. alarm cues novel tank behaviour plasticity novel odour uncertainty Figures Figure 1 Figure 2 1. Introduction Balancing predator avoidance with other essential activities, such as foraging, courtship, and territorial defense, is a constant challenge for prey animals (Johnson et al. 2013 ; Lima and Dill 1990 ; Preisser et al. 2005 ; Sih 1992 ). Navigating these trade-offs is crucial due to the unforgiving nature of predation, as a misjudgment can cost the prey its life (Johnson et al. 2013 ; Lima and Dill 1990 ). Behavioural plasticity allows prey to exhibit appropriate antipredator responses depending on the level of predation threat (Brown et al. 2006 ; Helfman and Winkelman 1997 ). Such plasticity enables prey to balance predator avoidance and other fitness-related activities (Brown 2003 ). For example, small reef fishes exhibited suppressed foraging behavior near potential predators (Catano et al. 2016 ). Individuals displaying suitable antipredator responses toward predators are more likely to survive (Blumstein et al. 2002 ). To respond to predators, prey must first be able to sense danger (Endler 1986 ). Prey depend on accessible public information (tactile, auditory, visual, or chemical) regarding local threats to perceive risk (Bouskila and Blumstein 1992 ; Chivers and Smith 1998 ; Endler 1986 ). Many aquatic species use chemical alarm cues as a reliable cue to perceive predation risk. Chemical alarm cues are substances released from prey tissue when a predator damages it during an attack (Ferrari et al. 2010 ). When nearby individuals detect conspecific alarm cues, they exhibit overt antipredator responses (e.g., in zebrafish; Attaran et al. 2019 ). In general, the intensity of such antipredator responses is known to match the intensity of the threat (i.e., threat-sensitivity)(Helfman 1989 ). According to the “dangerous niche hypothesis”, individuals from high-predation environments should exhibit strong anti-predator responses to novel cues, as novelty is likely dangerous (Greenberg 1990 ; Mettke-Hofmann et al. 2013 ). Such fear of novelty (i.e., neophobia) should increase the probability of survival if novel cues are indeed dangerous (Crane et al. 2020 ). Prey have been reported to show phenotypically-plastic neophobia toward cues such as odours from novel species that are potential predators (i.e., predator neophobia), as well as fear towards a novel environment where danger might be present (i.e., spatial neophobia) (Brown et al. 2013 ; Crane and Ferrari 2017 ). For example, juvenile convict cichlids, Amatitlania nigrofasciata , and wood frog tadpoles, Lithobates sylvaticus , that were repeatedly exposed to conspecific alarm cues over a period of a few days became neophobic toward odours from novel species (Brown et al. 2013 ). Similarly, in fathead minnows, Pimephales promelas , repeated exposures to conspecific alarm cues induced spatial neophobia (Crane et al. 2020 ). In the presence of an actual threat, such neophobia can decrease the chances of making costly decisions until more data is obtained (Elvidge et al. 2016 ). Hence, neophobia allows prey to increase their survival in encounters with a novel predator, as has been found in whitetail damselfish, Pomacentrus chrysurus , (Ferrari et al. 2015 ) and wood frog tadpoles (Crane et al. 2018). Zebrafish, Danio rerio , has been widely used as a model species across biological fields, including genetics, pharmacology, neuroscience, and developmental biology (Miller and Gerlai 2011 ; Petersen et al. 2022 ). Characteristics such as their easy maintenance in high density, ability to produce many offspring, and high genetic homology with humans have made zebrafish an ideal model organism for such research (Barbazuk et al. 2000 ; Lawrence 2007 ). While zebrafish have gained popularity as a model for behavioural studies (Barcellos et al. 2010 ; Gerlai 2010 ; Miller and Gerlai 2011 ; Orger and Polavieja 2017 ), neophobia in zebrafish had not been investigated until recently (Franks et al. 2022 ; Lucon-Xiccato et al. 2020 ; Quadros et al. 2019 ; Roy et al. 2017 ). Most of these studies tested baseline neophobic responses toward novel objects or a novel environment. To our knowledge, only one study has explored induced neophobia in zebrafish (Quadros et al. 2019 ). Following repeated exposures to alarm cues over seven days, two zebrafish populations ( wild-type and leopard ) showed anxiety-like behaviours in a novel tank (i.e., spatial neophobia) (Quadros et al. 2019 ). However, it is still unknown whether repeated exposures over few days would induce neophobia in zebrafish in response to a novel predator odour (i.e. predator neophobia). In this study, we performed two experiments to investigate the role of background risk in inducing both spatial and predator odour neophobia in zebrafish. For both experiments, we repeatedly exposed the zebrafish to either alarm cues to simulate a high-risk environment or a water control (low risk) over five days. One day later, we tested zebrafish fear responses in a novel tank (experiment 1) and in the presence of a novel predator odour (rainbow trout, Oncorhynchus mykiss ) (experiment 2). We hypothesized that high background risk would induce both spatial and predator neophobia in zebrafish, as previous study reported that zebrafish exhibit spatial neophobia following repeated exposure to risk (Quadros et al. 2019 ). Hence, we predicted that high-risk zebrafish would be less active and exploratory in the novel tank and when exposed to the novel odour. 2. Materials and methods Test species and maintenance We used 150 experimentally naïve adult zebrafish (male and female, 12 months old) from a stock colony housed at the R. J. F. Smith Centre for Aquatic Ecology at the University of Saskatchewan. Zebrafish were transferred into 25 tanks (2.8 L tank, six fish per tank) with flow-through water that was filtered and dechlorinated. This water (hereafter, ‘facility water’) had a total hardness of 150 mg/L, alkalinity of 120 mg/L, pH of 7.6-8, and a temperature of 24-27 o C. All sides of the tank except the front were covered with opaque plastic sheets to block visual stimuli from nearby tanks. We fed zebrafish twice daily with commercial flake food. To obtain a novel odour, we used four rainbow trout (fork length 15.0–18.5 cm) from a stock colony at the Toxicology Centre at the University of Saskatchewan. The trout were housed in a 600 L flow-through pool with facility water. We fed the rainbow trout daily with commercial trout pellets. All fish were provided with a 14:10 light-to-dark cycle. Cue collection Alarm cues : Following standard methods (Attaran et al. 2019 ), we obtained zebrafish alarm cues by euthanizing five donor individuals with a blow to the head followed by a collection of 5.74 cm 2 skin from the lateral sides of the body. We homogenized the skin in 200 ml of facility water and removed any remaining large particles by filtering the solution through a mesh (0.5 mm). The resulting solution was then diluted with water to reach a final concentration of -1 cm 2 per 40 L of water, which is known to elicit a significant antipredator response in zebrafish (Attaran et al. 2019 ). We then stored the final solution in 100 mL aliquots at 20 o C until it was thawed and used for the exposure treatment. Additionally, we froze 20 mL samples of facility water as a control. Novel odour To obtain a novel odour, we transferred the trout into individual 38 L tanks filled with clean facility water at a volume standardized for the size of the fish (50 mL/g of fish). The fish had been deprived of food for 48 hours beforehand to minimize diet cues (Ferrari et al. 2008 ; Scherer and Smee 2016 ). After 24 hours, tank water was collected and frozen in 600-ml aliquots until being thawed before use. Experimental overview: (i) Background risk exposure phase We exposed the zebrafish in groups of six in 2.8 L plastic tanks (housing tanks) equipped with an air stone. All sides of the tank except the front were covered with opaque plastic sheets to block visual stimuli from nearby tanks. A 150-cm injection hose, attached parallel to the air stone, facilitated the gentle introduction cues into the tank using a syringe (Crane et al. 2015 ; Ferrari and Chivers 2006a , 2006b ). Injections occurred three times a day (between 0800 and 1600 h) for five consecutive days with either 0.5 mL of conspecific alarm cues (high risk) or facility water (low risk). A complete water change was conducted one hour after the third exposure each day. Spatial neophobia (Experiment 1) : We assessed spatial neophobia in a novel environment 24 hours after the background exposure phase. This ‘novel tank test’ is an established paradigm to study spatial neophobia (Blaser and Rosemberg 2012 ; Cachat et al. 2010 ; Cachat et al. 2011 ; Salahinejad et al. 2022 ). The test tank differed in size (28.8 × 16.2 × 10 cm) and shape (rectangular) from the holding tanks. We used two 13-watt fluorescent bulbs to illuminate the tank from above. White plastic sheets covered all three sides of the tank except the front. Additionally, the tank was equipped with a plastic injection hose that ended at 1.5 cm below the water surface. We placed a single zebrafish into the test tank filled with tap water and acclimated it for 10 seconds. Following the acclimation, we recorded zebrafish behaviour for eight minutes using an HD webcam (C922x Pro Stream, Logitech, Lausanne, Switzerland). We used a short acclimation period to prevent individuals from becoming accustomed to the new tank. We tested a total of 60 individuals. Predator odour neophobia (Experiment 2) : To assess the response to a novel predator odour, we subjected zebrafish to the background risk exposure phase mentioned above, followed by testing individual behaviour in response to the novel predator odour (rainbow trout odour). One day after the last alarm cues exposure, we placed a single zebrafish in the novel tank for 30 minutes to allow it to habituate to the new environment. Previous studies have reported that a 30-minute period is enough for zebrafish to habituate in a novel tank (Raymond et al. 2012 ; Wong et al. 2010 ). We then began the predator neophobia testing trial by recording zebrafish in the tank for six minutes (i.e., the ‘pre-stimulus period’). Following the pre-stimulus period, we gently injected 10 mL of rainbow trout odour or facility water into the tank and recorded individual behaviour for another six minutes (the ‘post-stimulus period’). We tested a total of 80 individuals in this experiment. Quantification of Behaviour We analyzed the recorded videos using Ethovision XT (Noldus Info Tech., Wageningen, The Netherlands) as described by (Blaser and Gerlai 2006 ; Gerlai et al. 2008 ). Briefly, we used automatic tracking by the software to identify the subject within a defined arena (test tank area) at 10 Hz recording frequency (once every 0.1 s). As a result, 600 position x, y coordinate pairs per minute were recorded for each fish. Using the software, we defined three equal vertical layers (bottom, middle, and top) on the testing tank. We quantified the distance travelled, time spent in each layer, and latency to reach the top layer. Previous studies have reported that fearful zebrafish show reduced movement, more time spent in the bottom layer of the tank, less time spent in the top layer, and a longer latency to reach the top layer (Gerlai et al. 2000 ; Jesuthasan and Mathuru 2008 ; Pfeiffer 1977 ; Salahinejad et al. 2022 ). Statistical Analysis Spatial neophobia (Experiment 1) We performed a principal component analysis (PCA) to reduce the number of response variables by creating a composite fear response using the variables: total distance travelled, time spent in the bottom layer, time spent in the top layer, and latency to reach the top layer of the tank. The PCA used a covariance matrix. For the novel tank test, the PCA resulted in one axis (PC1) that explained 80.36% of the total variance (eigenvalue > 3). The scores loaded heavily on more distance traveled (0.96), less time spent in the bottom layer (-0.95), more time spent in the top layer (0.90), and a shorter latency to reach the top layer (-0.76). We multiplied PC1 by -1 for interpretability so that larger values would represent stronger fear responses. Hence, we refer to PC1 as the ‘fear responses’ hereafter. We then analyzed differences in PC1 between high- and low-risk zebrafish using a Type-I nested ANOVA, with the background risk treatment as a fixed factor and the exposure tank as a random factor, with fish nested within their exposure tanks. We therefore considered the tank, rather than the fish, the unit of replication. Predator odour neophobia (Experiment 2) Novel odour baseline test We did a PCA to reduce the number of variables to analyze the pre-stimulus baseline behaviour (i.e., before the novel odour was introduced). PC1 explained 58.99% of the total variance (eigenvalue > 2) and loaded heavily on less distance travelled (-0.43), more time spent in the bottom layer (0.92), less time spent in the top layer (-0.88), and a longer latency to reach the top layer (0.74) (Fig. 1). We analyzed this ‘baseline fear response’ (PC1) using a Type-I two-way nested ANOVA with the background risk treatment (high or low), the test cue (novel odour or water), and their interaction as fixed factors and the exposure tank as a random factor. This confirmed that the pre-stimulus fear response did not differ significantly across the treatments (all P ’s > 0.05). Novel odour response As zebrafish had similar baseline activity, we calculated the change (post-stimulus – pre-stimulus) in behavioural variables (distance travelled, time in the bottom layer, time in the top layer, and latency to reach the top layer) and included these variables in a PCA. PC1 explained 68.3% of the variance (eigenvalue ~ 3) and loaded on loaded heavily on less distance travelled (-0.59), more time spent in the bottom layer (0.96), less time spent in the top layer (-0.82), and a longer latency to reach the top layer (0.89). We then analyzed PC1 (i.e., the ‘change in fear response’) with a Type-I two-way nested ANOVA, including the background treatment (high risk or low risk), the test cue (novel odour or water) and their interaction as fixed factors, and exposure tank as a random factor. To further explore the interaction term, we split the data by the background treatment and used separate independent t-tests to compare responses to the test cues. All analyses were conducted in SPSS 26.0 with α = 0.05. 3. Results Spatial Neophobia (Experiment 1) We found a significant effect of background risk on fear responses of zebrafish in the novel tank, where individuals with repeated alarm cues exposure (high-risk) showed significantly higher fear responses compared to individuals exposed to water (low-risk) ( F 1,8 = 584.28, P < 0.001; Fig. 1). We did not find a significant effect of exposure tank on the fear response ( F 1,50 = 0.31, P = 0.96). Predator Neophobia (Experiment 2) : We found a significant interaction effect of background risk and test cue on the change in fear responses of zebrafish ( F 1,64 = 48.91, P < 0.001; Fig. 2 ). Individuals from high background risk showed a significantly higher fear responses when exposed to a novel predator odour compared to water ( t 31.93 = -8.78, P < 0.001; Fig. 2 ). However, low-risk zebrafish showed no significant fear responses when exposed to a novel predator odour or water ( t 31.43 = 0.71, P = 0.49; Fig. 2 ). We did not find a significant effect of background conditioning tanks on the change in fear response ( F 12,64 = 1.27, P = 0.26). 4. Discussion Our results suggest that repeated exposures to high predation risk can induce spatial and predator odour neophobia in zebrafish. Consistent with the “dangerous niche hypothesis” (Greenberg and Mettke-Hofmann 2001 ; Mettke-Hofmann et al. 2013 ), high-risk zebrafish showed reduced distance moved, increased time spent in the bottom layer, decreased time spent on the top layer, and delayed entry into the top layer of the novel tank, indicative of spatial neophobia. Furthermore, high-risk individuals displayed higher fear responses towards a novel predator odour, showing adaptive behaviour in response to potential threats from an unknown predator. Previous studies have also reported spatial and predator neophobia in other fish species (Elvidge et al. 2016 ; Feyten et al. 2019 ). Our results are consistent with the finding of Quadros et al. ( 2019 ), who reported anxiety-like behavior in zebrafish in a novel tank after repeated exposures to chemical alarm cues. Incomplete or partial information regarding predation risk may induce uncertainty in prey [reviewed by (Crane et al. 2023 )]. In our present studies, we repeatedly exposed the zebrafish to risk (conspecific alarm cue) without any predator cue. This lack of information about predation risk may have induced uncertainty about predator identity in zebrafish. When uncertain about the risk associated with a novel environment or cue, prey should exhibit neophobia (Elvidge et al. 2016 ). Although this neophobic response can incur significant energy costs, it can help the prey avoid risking its life in an unknown environment or cue (Ferrari et al. 2015 ). For instance, fathead minnows, Pimephales promelas , repeatedly exposed to chemical alarm cues without any predator cues showed neophobic predator responses in a novel environment. In experiment 1, when high-risk individuals were exposed to a novel tank, they showed antipredator responses to avoid unknown threats. Similarly, when high-risk individuals were exposed to a novel predator odour, they showed neophobic antipredator responses. Interestingly, in experiment 2, within a short time (30 minutes after introducing into the novel tank), there was no significant difference in antipredator responses between the high-risk and low-risk individuals. There could be two possible reasons behind this. Firstly, individuals may have stopped exhibiting antipredator responses without a negative reinforcement in the novel tank shortly after the initial introduction. Brown et al. (2015) reported that repeated exposures to a novel odour without any negative reinforcement allowed juvenile convict cichlids, Amatitlania nigrofasciata , to reduce neophobic responses quickly. Secondly, zebrafish are known for their robust habituation responses (Wong et al., 2010 ). The absence of negative reinforcement and fast habituation ability may have led to such a result. However, upon exposure to the novel predator odour, high-risk individuals again exhibited neophobic responses compared to low-risk individuals. It suggests that neophobia is a plastic response; individuals show fear in new environments or in response to new cues, but as they learn these are not threats, they stop showing costly fear responses. However, we did not record when zebrafish stopped exhibiting fear responses following the novel odour exposure. Future studies may compare the duration of antipredator responses between a novel environment and a novel cue. In our ever-changing world, species often find themselves in novel environments with unfamiliar predators. While exhibiting a neophobic response can undoubtedly confer survival benefits to prey, the "maladaptive defensive carry-over" concept suggests that an abrupt shift from a high-risk to a low-risk environment can result in unnecessary energy expenditure (Crane et al. 2020 ). In such cases, individuals with faster learning and memory acquisition ability can adjust to the new environment with appropriate behavioural plasticity. Only a few studies have investigated the role of background risk on prey's learning performance (Braithwaite and Brown 2004 ; Burns and Rodd 2008 ; Guido et al. 2017 ). More studies on the effects of neophobia on learning will allow us to predict which species will be well-adaptive or more vulnerable in the face of habitat shift. Declarations Competing Interests The authors declare no conflicts of interest. Ethical Statement Experiments were conducted in accordance with the University of Saskatchewan’s Committee on Animal Care and Supply (protocol 20190098). Following the experiments, we euthanized the zebrafish by rapid chilling. Author Contribution HT and MCOF designed the study. HT, AS, and AG conducted the experiments. HT performed formal analysis and wrote the manuscript. All the authors contributed to editing the manuscript. Acknowledgement This research was funded by The Natural Sciences and Engineering Research Council of Canada to M.C.O.F in the form of a Discovery Grant. Data Availability Behavioural data that support the findings of this study is provided as a supplementary information files References Attaran A, Salahinejad A, Crane AL, Niyogi S, Chivers DP (2019) Chronic exposure to dietary selenomethionine dysregulates the genes involved in serotonergic neurotransmission and alters social and antipredator behaviours in zebrafish ( Danio rerio ). Environ Pollut 246:837–844 Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Johnson SL (2000) The syntenic relationship of the zebrafish and human genomes. 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Am Nat 139(5):1052–1069 Wong K, Elegante M, Bartels B, Elkhayat S, Tien D, Roy S, Grimes C (2010) Analyzing habituation responses to novelty in zebrafish ( Danio rerio ). Behav Brain Res 208(2):450–457 Additional Declarations No competing interests reported. Supplementary Files Datafile.xlsx Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 23 Oct, 2024 Read the published version in Animal Cognition → Version 1 posted Editorial decision: Revision requested 25 Jul, 2024 Reviews received at journal 30 Jun, 2024 Reviews received at journal 25 Jun, 2024 Reviewers agreed at journal 05 Jun, 2024 Reviewers agreed at journal 31 May, 2024 Reviewers invited by journal 27 May, 2024 Editor assigned by journal 27 May, 2024 Submission checks completed at journal 27 May, 2024 First submitted to journal 25 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4477880","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311085507,"identity":"540697c7-0a1a-4a88-b50e-f9e71dc6ac66","order_by":0,"name":"Himal Thapa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACA2Yg8YCBIQFIMT4AiUgQpSWBwQCkhdmAOC0MCC1sEkRpMWfnTvyQwPAnj39287Nqnj929pINzA8/4NNi2cy7WQJoS7HEnWNmt3nbkhNnM7AZ47XJ4DDvBpCWxIYbCUAtDcwJcgw8+B0H1LL5B0jL/Bvp34p5/tTbA7Uw/yCgZRvYlg03csyYedgOM85m4GEjZMs2iwQD48SNd84US85tO544s5nNzAKvlvNnN9/4UCGXOO92+8YPb/5U20scb358A58WqEYGpPhgJqweCghG4SgYBaNgFIxYAAAED0aWXLfoKAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":true,"prefix":"","firstName":"Himal","middleName":"","lastName":"Thapa","suffix":""},{"id":311085508,"identity":"45bc6e32-94ed-486e-9c24-c1154971cf48","order_by":1,"name":"Arash Salahinejad","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Arash","middleName":"","lastName":"Salahinejad","suffix":""},{"id":311085510,"identity":"809ca846-4c69-4654-8422-4fc87dac56b2","order_by":2,"name":"Adam L. Crane","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"L.","lastName":"Crane","suffix":""},{"id":311085512,"identity":"80919261-f9ea-4d08-848c-6b38ccd60e1b","order_by":3,"name":"Ahmad Ghobeishavi","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Ghobeishavi","suffix":""},{"id":311085513,"identity":"92dac1a4-5d56-4d2b-b82e-42d9c325a24d","order_by":4,"name":"Maud C. O. Ferrari","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Maud","middleName":"C. O.","lastName":"Ferrari","suffix":""}],"badges":[],"createdAt":"2024-05-25 19:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4477880/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4477880/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10071-024-01908-z","type":"published","date":"2024-10-23T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58171163,"identity":"b047a9c9-49c9-4e88-b9be-cc2edc032f01","added_by":"auto","created_at":"2024-06-12 03:44:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118100,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±SE) a) fear response (PC1), b) total distance travelled, c) Time spent in bottom zone, d) latency to top zone, and e) time spent in top zone in a novel environment by zebrafish from either a high-risk (alarm cue) or low-risk (water) background\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4477880/v1/5f38d3f8c71628f41b413706.png"},{"id":58170014,"identity":"dfb7d795-778d-48fb-9949-c9bfa065d6c3","added_by":"auto","created_at":"2024-06-12 03:36:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112902,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±SE) a) fear response (PC1), b) total distance travelled, c) time spent in bottom zone, d) time spent in top zone, and e) change in latency to top zone by zebrafish from high-risk (alarm cue) or low-risk (water) backgrounds when tested for a response to a novel odour vs. a water control. White bars represent water cue and grey bars represent novel odour\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4477880/v1/85c3ac62f89b1cdd0b1be49c.png"},{"id":67682772,"identity":"570703c9-db07-4859-9c1b-7f2c3fb6e9dd","added_by":"auto","created_at":"2024-10-28 16:15:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":522709,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4477880/v1/ba69675e-5d9c-4a66-9573-87fb8ff71694.pdf"},{"id":58170016,"identity":"02b3ade1-7e22-4d5b-8f84-27edc58fade3","added_by":"auto","created_at":"2024-06-12 03:36:05","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26347,"visible":true,"origin":"","legend":"","description":"","filename":"Datafile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4477880/v1/0a6f1eb4fb905b056b5e1981.xlsx"},{"id":58170017,"identity":"2c669c32-cb97-4e32-8c95-bc7ed36c74ba","added_by":"auto","created_at":"2024-06-12 03:36:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":53898,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4477880/v1/ebdc3f6a94ec9a47a54b0cb9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Background predation risk induces neophobia in zebrafish","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBalancing predator avoidance with other essential activities, such as foraging, courtship, and territorial defense, is a constant challenge for prey animals (Johnson et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lima and Dill \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Preisser et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sih \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Navigating these trade-offs is crucial due to the unforgiving nature of predation, as a misjudgment can cost the prey its life (Johnson et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lima and Dill \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Behavioural plasticity allows prey to exhibit appropriate antipredator responses depending on the level of predation threat (Brown et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Helfman and Winkelman \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Such plasticity enables prey to balance predator avoidance and other fitness-related activities (Brown \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For example, small reef fishes exhibited suppressed foraging behavior near potential predators (Catano et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Individuals displaying suitable antipredator responses toward predators are more likely to survive (Blumstein et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo respond to predators, prey must first be able to sense danger (Endler \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Prey depend on accessible public information (tactile, auditory, visual, or chemical) regarding local threats to perceive risk (Bouskila and Blumstein \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Chivers and Smith \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Endler \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Many aquatic species use chemical alarm cues as a reliable cue to perceive predation risk. Chemical alarm cues are substances released from prey tissue when a predator damages it during an attack (Ferrari et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). When nearby individuals detect conspecific alarm cues, they exhibit overt antipredator responses (e.g., in zebrafish; Attaran et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e ). In general, the intensity of such antipredator responses is known to match the intensity of the threat (i.e., threat-sensitivity)(Helfman \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the \u0026ldquo;dangerous niche hypothesis\u0026rdquo;, individuals from high-predation environments should exhibit strong anti-predator responses to novel cues, as novelty is likely dangerous (Greenberg \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Mettke-Hofmann et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Such fear of novelty (i.e., neophobia) should increase the probability of survival if novel cues are indeed dangerous (Crane et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Prey have been reported to show phenotypically-plastic neophobia toward cues such as odours from novel species that are potential predators (i.e., predator neophobia), as well as fear towards a novel environment where danger might be present (i.e., spatial neophobia) (Brown et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Crane and Ferrari \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, juvenile convict cichlids, \u003cem\u003eAmatitlania nigrofasciata\u003c/em\u003e, and wood frog tadpoles, \u003cem\u003eLithobates sylvaticus\u003c/em\u003e, that were repeatedly exposed to conspecific alarm cues over a period of a few days became neophobic toward odours from novel species (Brown et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, in fathead minnows, \u003cem\u003ePimephales promelas\u003c/em\u003e, repeated exposures to conspecific alarm cues induced spatial neophobia (Crane et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the presence of an actual threat, such neophobia can decrease the chances of making costly decisions until more data is obtained (Elvidge et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hence, neophobia allows prey to increase their survival in encounters with a novel predator, as has been found in whitetail damselfish, \u003cem\u003ePomacentrus chrysurus\u003c/em\u003e, (Ferrari et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and wood frog tadpoles (Crane et al. 2018).\u003c/p\u003e \u003cp\u003eZebrafish, \u003cem\u003eDanio rerio\u003c/em\u003e, has been widely used as a model species across biological fields, including genetics, pharmacology, neuroscience, and developmental biology (Miller and Gerlai \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Petersen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Characteristics such as their easy maintenance in high density, ability to produce many offspring, and high genetic homology with humans have made zebrafish an ideal model organism for such research (Barbazuk et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Lawrence \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). While zebrafish have gained popularity as a model for behavioural studies (Barcellos et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gerlai \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Miller and Gerlai \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Orger and Polavieja \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), neophobia in zebrafish had not been investigated until recently (Franks et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lucon-Xiccato et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Quadros et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Most of these studies tested baseline neophobic responses toward novel objects or a novel environment. To our knowledge, only one study has explored induced neophobia in zebrafish (Quadros et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Following repeated exposures to alarm cues over seven days, two zebrafish populations (\u003cem\u003ewild-type\u003c/em\u003e and \u003cem\u003eleopard\u003c/em\u003e) showed anxiety-like behaviours in a novel tank (i.e., spatial neophobia) (Quadros et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, it is still unknown whether repeated exposures over few days would induce neophobia in zebrafish in response to a novel predator odour (i.e. predator neophobia).\u003c/p\u003e \u003cp\u003eIn this study, we performed two experiments to investigate the role of background risk in inducing both spatial and predator odour neophobia in zebrafish. For both experiments, we repeatedly exposed the zebrafish to either alarm cues to simulate a high-risk environment or a water control (low risk) over five days. One day later, we tested zebrafish fear responses in a novel tank (experiment 1) and in the presence of a novel predator odour (rainbow trout, \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) (experiment 2). We hypothesized that high background risk would induce both spatial and predator neophobia in zebrafish, as previous study reported that zebrafish exhibit spatial neophobia following repeated exposure to risk (Quadros et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hence, we predicted that high-risk zebrafish would be less active and exploratory in the novel tank and when exposed to the novel odour.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTest species and maintenance\u003c/p\u003e \u003cp\u003eWe used 150 experimentally naïve adult zebrafish (male and female, 12 months old) from a stock colony housed at the R. J. F. Smith Centre for Aquatic Ecology at the University of Saskatchewan. Zebrafish were transferred into 25 tanks (2.8 L tank, six fish per tank) with flow-through water that was filtered and dechlorinated. This water (hereafter, ‘facility water’) had a total hardness of 150 mg/L, alkalinity of 120 mg/L, pH of 7.6-8, and a temperature of 24-27\u003csup\u003eo\u003c/sup\u003e C. All sides of the tank except the front were covered with opaque plastic sheets to block visual stimuli from nearby tanks. We fed zebrafish twice daily with commercial flake food.\u003c/p\u003e \u003cp\u003eTo obtain a novel odour, we used four rainbow trout (fork length 15.0–18.5 cm) from a stock colony at the Toxicology Centre at the University of Saskatchewan. The trout were housed in a 600 L flow-through pool with facility water. We fed the rainbow trout daily with commercial trout pellets. All fish were provided with a 14:10 light-to-dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eCue collection\u003c/p\u003e \u003cp\u003e \u003cem\u003eAlarm cues\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eFollowing standard methods (Attaran et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), we obtained zebrafish alarm cues by euthanizing five donor individuals with a blow to the head followed by a collection of 5.74 cm\u003csup\u003e2\u003c/sup\u003e skin from the lateral sides of the body. We homogenized the skin in 200 ml of facility water and removed any remaining large particles by filtering the solution through a mesh (0.5 mm). The resulting solution was then diluted with water to reach a final concentration of -1 cm\u003csup\u003e2\u003c/sup\u003e per 40 L of water, which is known to elicit a significant antipredator response in zebrafish (Attaran et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We then stored the final solution in 100 mL aliquots at 20\u003csup\u003eo\u003c/sup\u003eC until it was thawed and used for the exposure treatment. Additionally, we froze 20 mL samples of facility water as a control.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNovel odour\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo obtain a novel odour, we transferred the trout into individual 38 L tanks filled with clean facility water at a volume standardized for the size of the fish (50 mL/g of fish). The fish had been deprived of food for 48 hours beforehand to minimize diet cues (Ferrari et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Scherer and Smee \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). After 24 hours, tank water was collected and frozen in 600-ml aliquots until being thawed before use.\u003c/p\u003e \u003cp\u003eExperimental overview:\u003c/p\u003e \u003cp\u003e \u003cem\u003e(i) Background risk exposure phase\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe exposed the zebrafish in groups of six in 2.8 L plastic tanks (housing tanks) equipped with an air stone. All sides of the tank except the front were covered with opaque plastic sheets to block visual stimuli from nearby tanks. A 150-cm injection hose, attached parallel to the air stone, facilitated the gentle introduction cues into the tank using a syringe (Crane et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ferrari and Chivers \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e). Injections occurred three times a day (between 0800 and 1600 h) for five consecutive days with either 0.5 mL of conspecific alarm cues (high risk) or facility water (low risk). A complete water change was conducted one hour after the third exposure each day.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSpatial neophobia (Experiment 1)\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eWe assessed spatial neophobia in a novel environment 24 hours after the background exposure phase. This ‘novel tank test’ is an established paradigm to study spatial neophobia (Blaser and Rosemberg \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cachat et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cachat et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Salahinejad et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The test tank differed in size (28.8 × 16.2 × 10 cm) and shape (rectangular) from the holding tanks. We used two 13-watt fluorescent bulbs to illuminate the tank from above. White plastic sheets covered all three sides of the tank except the front. Additionally, the tank was equipped with a plastic injection hose that ended at 1.5 cm below the water surface. We placed a single zebrafish into the test tank filled with tap water and acclimated it for 10 seconds. Following the acclimation, we recorded zebrafish behaviour for eight minutes using an HD webcam (C922x Pro Stream, Logitech, Lausanne, Switzerland). We used a short acclimation period to prevent individuals from becoming accustomed to the new tank. We tested a total of 60 individuals.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePredator odour neophobia (Experiment 2)\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eTo assess the response to a novel predator odour, we subjected zebrafish to the background risk exposure phase mentioned above, followed by testing individual behaviour in response to the novel predator odour (rainbow trout odour). One day after the last alarm cues exposure, we placed a single zebrafish in the novel tank for 30 minutes to allow it to habituate to the new environment. Previous studies have reported that a 30-minute period is enough for zebrafish to habituate in a novel tank (Raymond et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wong et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We then began the predator neophobia testing trial by recording zebrafish in the tank for six minutes (i.e., the ‘pre-stimulus period’). Following the pre-stimulus period, we gently injected 10 mL of rainbow trout odour or facility water into the tank and recorded individual behaviour for another six minutes (the ‘post-stimulus period’). We tested a total of 80 individuals in this experiment.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantification of Behaviour\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the recorded videos using Ethovision XT (Noldus Info Tech., Wageningen, The Netherlands) as described by (Blaser and Gerlai \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gerlai et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Briefly, we used automatic tracking by the software to identify the subject within a defined arena (test tank area) at 10 Hz recording frequency (once every 0.1 s). As a result, 600 position x, y coordinate pairs per minute were recorded for each fish. Using the software, we defined three equal vertical layers (bottom, middle, and top) on the testing tank. We quantified the distance travelled, time spent in each layer, and latency to reach the top layer. Previous studies have reported that fearful zebrafish show reduced movement, more time spent in the bottom layer of the tank, less time spent in the top layer, and a longer latency to reach the top layer (Gerlai et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Jesuthasan and Mathuru \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pfeiffer \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Salahinejad et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStatistical Analysis\u003c/p\u003e \u003cp\u003e \u003cem\u003eSpatial neophobia (Experiment 1)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe performed a principal component analysis (PCA) to reduce the number of response variables by creating a composite fear response using the variables: total distance travelled, time spent in the bottom layer, time spent in the top layer, and latency to reach the top layer of the tank. The PCA used a covariance matrix. For the novel tank test, the PCA resulted in one axis (PC1) that explained 80.36% of the total variance (eigenvalue \u0026gt; 3). The scores loaded heavily on more distance traveled (0.96), less time spent in the bottom layer (-0.95), more time spent in the top layer (0.90), and a shorter latency to reach the top layer (-0.76). We multiplied PC1 by -1 for interpretability so that larger values would represent stronger fear responses. Hence, we refer to PC1 as the ‘fear responses’ hereafter. We then analyzed differences in PC1 between high- and low-risk zebrafish using a Type-I nested ANOVA, with the background risk treatment as a fixed factor and the exposure tank as a random factor, with fish nested within their exposure tanks. We therefore considered the tank, rather than the fish, the unit of replication.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePredator odour neophobia (Experiment 2)\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eNovel odour baseline test\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe did a PCA to reduce the number of variables to analyze the pre-stimulus baseline behaviour (i.e., before the novel odour was introduced). PC1 explained 58.99% of the total variance (eigenvalue \u0026gt; 2) and loaded heavily on less distance travelled (-0.43), more time spent in the bottom layer (0.92), less time spent in the top layer (-0.88), and a longer latency to reach the top layer (0.74) (Fig.\u0026nbsp;1). We analyzed this ‘baseline fear response’ (PC1) using a Type-I two-way nested ANOVA with the background risk treatment (high or low), the test cue (novel odour or water), and their interaction as fixed factors and the exposure tank as a random factor. This confirmed that the pre-stimulus fear response did not differ significantly across the treatments (all \u003cem\u003eP\u003c/em\u003e’s \u0026gt; 0.05).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNovel odour response\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAs zebrafish had similar baseline activity, we calculated the change (post-stimulus – pre-stimulus) in behavioural variables (distance travelled, time in the bottom layer, time in the top layer, and latency to reach the top layer) and included these variables in a PCA. PC1 explained 68.3% of the variance (eigenvalue ~ 3) and loaded on loaded heavily on less distance travelled (-0.59), more time spent in the bottom layer (0.96), less time spent in the top layer (-0.82), and a longer latency to reach the top layer (0.89). We then analyzed PC1 (i.e., the ‘change in fear response’) with a Type-I two-way nested ANOVA, including the background treatment (high risk or low risk), the test cue (novel odour or water) and their interaction as fixed factors, and exposure tank as a random factor. To further explore the interaction term, we split the data by the background treatment and used separate independent t-tests to compare responses to the test cues. All analyses were conducted in SPSS 26.0 with α = 0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"3. Results","content":"\u003cp\u003e \u003cem\u003eSpatial Neophobia (Experiment 1)\u003c/em\u003e \u003c/p\u003e\u003cp\u003eWe found a significant effect of background risk on fear responses of zebrafish in the novel tank, where individuals with repeated alarm cues exposure (high-risk) showed significantly higher fear responses compared to individuals exposed to water (low-risk) (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,8\u003c/sub\u003e = 584.28, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Fig.\u0026nbsp;1). We did not find a significant effect of exposure tank on the fear response (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,50\u003c/sub\u003e = 0.31, \u003cem\u003eP\u003c/em\u003e = 0.96).\u003c/p\u003e\u003cp\u003e \u003cem\u003ePredator Neophobia (Experiment 2)\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eWe found a significant interaction effect of background risk and test cue on the change in fear responses of zebrafish (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,64\u003c/sub\u003e = 48.91, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Individuals from high background risk showed a significantly higher fear responses when exposed to a novel predator odour compared to water (\u003cem\u003et\u003c/em\u003e\u003csub\u003e31.93\u003c/sub\u003e = -8.78, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, low-risk zebrafish showed no significant fear responses when exposed to a novel predator odour or water (\u003cem\u003et\u003c/em\u003e\u003csub\u003e31.43\u003c/sub\u003e = 0.71, \u003cem\u003eP\u003c/em\u003e = 0.49; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We did not find a significant effect of background conditioning tanks on the change in fear response (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e12,64\u003c/sub\u003e = 1.27, \u003cem\u003eP\u003c/em\u003e = 0.26).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results suggest that repeated exposures to high predation risk can induce spatial and predator odour neophobia in zebrafish. Consistent with the “dangerous niche hypothesis” (Greenberg and Mettke-Hofmann \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Mettke-Hofmann et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), high-risk zebrafish showed reduced distance moved, increased time spent in the bottom layer, decreased time spent on the top layer, and delayed entry into the top layer of the novel tank, indicative of spatial neophobia. Furthermore, high-risk individuals displayed higher fear responses towards a novel predator odour, showing adaptive behaviour in response to potential threats from an unknown predator. Previous studies have also reported spatial and predator neophobia in other fish species (Elvidge et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Feyten et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our results are consistent with the finding of Quadros et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who reported anxiety-like behavior in zebrafish in a novel tank after repeated exposures to chemical alarm cues.\u003c/p\u003e\u003cp\u003eIncomplete or partial information regarding predation risk may induce uncertainty in prey [reviewed by (Crane et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)]. In our present studies, we repeatedly exposed the zebrafish to risk (conspecific alarm cue) without any predator cue. This lack of information about predation risk may have induced uncertainty about predator identity in zebrafish. When uncertain about the risk associated with a novel environment or cue, prey should exhibit neophobia (Elvidge et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although this neophobic response can incur significant energy costs, it can help the prey avoid risking its life in an unknown environment or cue (Ferrari et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, fathead minnows, \u003cem\u003ePimephales promelas\u003c/em\u003e, repeatedly exposed to chemical alarm cues without any predator cues showed neophobic predator responses in a novel environment. In experiment 1, when high-risk individuals were exposed to a novel tank, they showed antipredator responses to avoid unknown threats. Similarly, when high-risk individuals were exposed to a novel predator odour, they showed neophobic antipredator responses.\u003c/p\u003e\u003cp\u003eInterestingly, in experiment 2, within a short time (30 minutes after introducing into the novel tank), there was no significant difference in antipredator responses between the high-risk and low-risk individuals. There could be two possible reasons behind this. Firstly, individuals may have stopped exhibiting antipredator responses without a negative reinforcement in the novel tank shortly after the initial introduction. Brown et al. (2015) reported that repeated exposures to a novel odour without any negative reinforcement allowed juvenile convict cichlids, \u003cem\u003eAmatitlania nigrofasciata\u003c/em\u003e, to reduce neophobic responses quickly. Secondly, zebrafish are known for their robust habituation responses (Wong et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The absence of negative reinforcement and fast habituation ability may have led to such a result. However, upon exposure to the novel predator odour, high-risk individuals again exhibited neophobic responses compared to low-risk individuals. It suggests that neophobia is a plastic response; individuals show fear in new environments or in response to new cues, but as they learn these are not threats, they stop showing costly fear responses. However, we did not record when zebrafish stopped exhibiting fear responses following the novel odour exposure. Future studies may compare the duration of antipredator responses between a novel environment and a novel cue.\u003c/p\u003e\u003cp\u003eIn our ever-changing world, species often find themselves in novel environments with unfamiliar predators. While exhibiting a neophobic response can undoubtedly confer survival benefits to prey, the \"maladaptive defensive carry-over\" concept suggests that an abrupt shift from a high-risk to a low-risk environment can result in unnecessary energy expenditure (Crane et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In such cases, individuals with faster learning and memory acquisition ability can adjust to the new environment with appropriate behavioural plasticity. Only a few studies have investigated the role of background risk on prey's learning performance (Braithwaite and Brown \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Burns and Rodd \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Guido et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). More studies on the effects of neophobia on learning will allow us to predict which species will be well-adaptive or more vulnerable in the face of habitat shift.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eEthical Statement\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;Experiments were conducted in accordance with the University of Saskatchewan\u0026rsquo;s Committee on Animal Care and Supply (protocol 20190098). Following the experiments, we euthanized the zebrafish by rapid chilling.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHT and MCOF designed the study. HT, AS, and AG conducted the experiments. HT performed formal analysis and wrote the manuscript. All the authors contributed to editing the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was funded by The Natural Sciences and Engineering Research Council of Canada to M.C.O.F in the form of a Discovery Grant.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eBehavioural data that support the findings of this study is provided as a supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAttaran A, Salahinejad A, Crane AL, Niyogi S, Chivers DP (2019) Chronic exposure to dietary selenomethionine dysregulates the genes involved in serotonergic neurotransmission and alters social and antipredator behaviours in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). 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Behav Brain Res 208(2):450\u0026ndash;457\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"animal-cognition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anco","sideBox":"Learn more about [Animal Cognition](http://link.springer.com/journal/10071)","snPcode":"10071","submissionUrl":"https://submission.nature.com/new-submission/10071/3","title":"Animal Cognition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"alarm cues, novel tank, behaviour plasticity, novel odour, uncertainty","lastPublishedDoi":"10.21203/rs.3.rs-4477880/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4477880/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrey face a major challenge in balancing predator avoidance with other essential activities. In environments with high risk, prey may exhibit neophobia (fear of novelty) due to the increased likelihood of novel stimuli being dangerous. The zebrafish, \u003cem\u003eDanio rerio\u003c/em\u003e, is an established model organism for many scientific studies. Although spatial and object neophobia in zebrafish have received previous attention, little is known about the role of background risk in inducing neophobia in zebrafish. Here, we present two experiments using zebrafish to explore whether background predation risk can induce fear in a novel environment and when exposed to a novel odour. Over five days, we repeatedly exposed zebrafish to either high background risk in the form of chemical alarm cues (i.e., injured conspecific cues that indicate a predator attack) or a low-risk water control stimulus. When tested in a novel spatial environment, zebrafish exposed to high predation risk displayed fear responses (reduced activity and bottom time) compared to their low-risk counterparts. Moreover, high-risk individuals exhibited fear responses toward a novel odour, unlike low-risk individuals. These results reveal that short-term repeated exposures to high risk can induce neophobia in zebrafish.\u003c/p\u003e","manuscriptTitle":"Background predation risk induces neophobia in zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 03:36:01","doi":"10.21203/rs.3.rs-4477880/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-25T14:53:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-01T01:02:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-25T20:25:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145772626415587716150805734049908336696","date":"2024-06-05T22:57:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59296108528820473230006867054259208072","date":"2024-05-31T12:38:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-28T02:05:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-27T22:54:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-27T14:46:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Animal Cognition","date":"2024-05-25T18:56:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"animal-cognition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anco","sideBox":"Learn more about [Animal Cognition](http://link.springer.com/journal/10071)","snPcode":"10071","submissionUrl":"https://submission.nature.com/new-submission/10071/3","title":"Animal Cognition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"59d10f30-db5e-48a4-a244-a803f7f96dbc","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:11:30+00:00","versionOfRecord":{"articleIdentity":"rs-4477880","link":"https://doi.org/10.1007/s10071-024-01908-z","journal":{"identity":"animal-cognition","isVorOnly":false,"title":"Animal Cognition"},"publishedOn":"2024-10-23 15:57:57","publishedOnDateReadable":"October 23rd, 2024"},"versionCreatedAt":"2024-06-12 03:36:01","video":"","vorDoi":"10.1007/s10071-024-01908-z","vorDoiUrl":"https://doi.org/10.1007/s10071-024-01908-z","workflowStages":[]},"version":"v1","identity":"rs-4477880","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4477880","identity":"rs-4477880","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}