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Alarm cue-mediated response and learning in zebrafish larvae
Behavioural Brain Research 380 (2020) 112446
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Research report
Alarm cue-mediated response and learning in zebrafish larvae a,
a
b
Tyrone Lucon-Xiccato *, Giuseppe Di Mauro , Angelo Bisazza , Cristiano Bertolucci a b
T a
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Dipartimento di Psicologia Generale, Università di Padova, Padova, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Alarm substances Fish behaviour Fish cognition Predator recognition Learning
We investigated the behavioural and learning response of zebrafish larvae to chemicals released by injured conspecifics (the alarm cue). Many aquatic vertebrates and invertebrates exhibit an innate antipredator response to alarm cues because in nature, they reliably indicate the presence of predators. Likewise, when an individual simultaneously perceives a novel odour and alarm cue, it learns to recognise the novel odour as a predator odour. Alarm cue-mediated behavioural response and learning have been reported in some fish and amphibians during early ontogeny, but in zebrafish, they have been described only for adults. In this study, we demonstrated that zebrafish at 12 and 24 days post fertilization exhibited reduced activity when exposed to alarm cue obtained by homogenised larvae of the same age, with this response being greater for the older zebrafish. In addition, we showed that 24-dpf zebrafish conditioned with alarm cue plus a novel odour learned to recognise the novel odour as a threat and responded to it with antipredator behaviour. The innate behavioural response and the learned response after conditioning may be used to develop paradigms with which to study anxiety, fear, stress, learning and memory in zebrafish larvae.
1. Introduction
species revealed that even their ink evokes alarm response [19]. Alarm cues are considered by most authors a mixture of different substances released upon damage that fish and other aquatic organisms use both individually, and collectively to identify predation risk (reviewed in Chivers et al. [20]). The zebrafish is also increasingly used for research on learning and memory [21–23], and alarm cues might be useful in this regard. Indeed, adult zebrafish, and other aquatic organisms, exploit alarm cue to learn about novel predators [24,25]. When a prey perceives alarm cue paired with a novel odour, it learns to recognise the novel odour as a threat. The conditioned response consists of antipredator behaviour (e.g., decreased activity) when exposed again to the conditioned cue (i.e., the novel odour), even without alarm cue [8–10]. This association between alarm cue and novel odour is extremely robust and occurs at the first presentation of the stimuli [10]. Intuitively, selection has favoured evolution of this one-trial learning mechanism to allow predator recognition after a single exposure, thereby increasing the prey’s chances of survival in future encounters [10]. Translational research is often interested in the use of larvae (i.e., age < 30 days post fertilisation, dpf) rather than of adult zebrafish [26]. Larvae can be collected in large numbers and tested early, given their quick development, making them particularly suitable to largescale screenings of drugs and genotypes. The innate behavioural
The zebrafish is gaining increasing importance as model for fear, stress and anxiety disorders [1,2]. For this reason, several behavioural paradigms have been developed (e.g. Hope et al. [3], Maximino et al. [4,5]), some of which exploit the innate response to alarm cues [6,7]. Alarm cues are substances contained in the skin and other tissues of many aquatic species [8–10]. When a prey is captured by a predator, the former’s damaged tissues release alarm cue in the water, which conspecifics can perceive via the olfactory system and use as a signal of danger to perform antipredator behaviours [8–10]. Adult zebrafish respond to alarm cues with typical antipredator behaviours, such as hiding or freezing, which cause a marked decrease in the locomotor activity [7]. Despite alarm cues having being studied for almost one century [11], their exact composition is unresolved. A line of evidence has suggested that hypoxanthine-3 N-oxide is the main substance causing antipredator response in fish [12,13], including in zebrafish [14]. However, this substance was not reliably detected in fish skin [15]. A recent biochemical fractionation study on zebrafish identified glycosaminoglycan chondroitin as the active substance [16]. Barreto and colleagues [17] reported that conspecific blood also evokes alarm response in Nile Tilapia. The alarm-cue effect of blood was detected also by studies on crustaceans [18], whereas a study on a cephalopod
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Corresponding author at: Department of Life Sciences and Biotechnology, Via L. Borsari 46, 44121, Ferrara, Italy. E-mail address: [email protected] (T. Lucon-Xiccato).
https://doi.org/10.1016/j.bbr.2019.112446 Received 2 July 2019; Received in revised form 18 December 2019; Accepted 19 December 2019 Available online 20 December 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Brain Research 380 (2020) 112446
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2. Materials and methods
response to alarm cue and the learned response to stimuli conditioned with alarm cue could be ideal for studying anxiety, fear and stress responses, as well as learning and memory, in zebrafish larvae. However, to the best of our knowledge, larvae’s behavioural responses to alarm cue have not been used as a research model. A possible explanation is that an early study has reported the onset of zebrafish’s alarm cue response at the age of 48–52 dpf [27]. One may argue that this result seems somehow counterintuitive. Being able to respond to and recognise predators should substantially increase the chance of survival for zebrafish larvae, as reported in other species [28]. Therefore, there should be strong selection for early development of this ability. In line with this idea, response to alarm cues has been consistently documented in juvenile fish and amphibians, even at the embryonic stage [29–34]. For example, Atherton and McCormick [29] monitored the heart beating of embryonic cinnamon clownfish, Amphiprion melanopus, exposed to conspecific alarm cue, finding significant increase in beating rates. This result was interpreted as evidence that cinnamon clownfish embryos can recognise alarm cue. Detecting zebrafish larvae’s responses to alarm cue may be difficult because of methodological issues. Larvae might respond differently or more subtly to alarm cues compared to adults; therefore, most of the methodologies used for adults might not be adequate for larvae. Another potential confound is that, in the early study, zebrafish larvae were exposed to the alarm cues of older conspecifics [27]. In two fish species—brook char, Salvelinus fontinalis, and spiny chromis, Acanthochromis polyacanthus—individuals can distinguish the age of alarm cue donors and preferentially respond to the alarm cue of individuals of the same age [35,36]. Because size increases with age in most of fish species, alarm cue from larger or smaller conspecifics may indicate the presence of a predator that is not dangerous because it preys on individuals of a different age class. It is therefore possible that zebrafish larvae do not respond to the alarm cue of older conspecifics because it indicates the presence of a predator species that preys on older conspecifics and does not represent an immediate threat. In experiment 1, we tested whether zebrafish larvae respond to alarm cue from same-age conspecifics. To detect the larvae’s response, we measured activity reduction and thigmotaxis using an automatic high-throughput tracking system, which was expected to improve the detection of the larvae’s behavioural responses. We compared three groups of larvae: one group exposed to alarm cue; one group exposed to water as a control, to ensure that the response in the alarm cue-exposed group was not due to the experimental manipulation; and one group exposed to fish odour as a further control for responses due to general fish odours or novel odours. We performed this experiment using subjects at 12 and 24 dpf. In experiment 2, we investigated whether larvae can use alarm cue to learn to recognise a novel odour as a threat. First, we conditioned 24dpf zebrafish using alarm cue paired with the odour of a novel, unfamiliar fish species to simulate a predator. After 6 h, we tested the larvae’s conditioned response to the fish odour alone; we also observed the response of a group of larvae exposed to water plus fish odour as control. Following a commonly-used approach [10], we assessed learning exploiting the antipredator response of larvae, which was identified in experiment 1 (i.e., activity reduction). In case of alarm cuemediated learning, we expected that the group conditioned with alarm cues and fish odour would thereafter show a significantly stronger decrease in activity when exposed to fish odour, as compared to the control group pseudoconditioned with water. Because we suspected that a carryover effect had occurred in experiment 2, causing fish exposed to alarm cues to show reduced baseline activity, we performed experiment 3 with a larger interval (24 h) between the conditioning phase and the test phase.
2.1. Animal husbandry We performed our experiments using larvae of wild-type zebrafish. The adults used for reproduction were maintained according to standard protocols in our laboratory at the University of Ferrara. Briefly, maintenance 200-L glass tanks housed mixed sex-groups with approximately 40 individuals. A heater kept the water temperature at 28 ± 1 °C, and illumination was provided with a 14:10 LD cycle. We fed the animals live brine shrimp nauplii (Ocean Nutrition, USA) and commercial flakes (Sera GmbH, D) twice per day. For reproduction, we transferred pairs of adult zebrafish into a breeding cage (Techniplast, I) in the late afternoon. We kept the two breeders divided by a transparent partition until light on of the following day, after which we removed the partition and the fish could spawn. We immediately collected the embryos and raised them in a Petri dish filled with E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSO4; 1 % of methylene blue). When they reached the age of 5 dpf, we moved the larvae into small glass tanks (20 × 15 cm, height 10 cm) filled with 2 cm of water (60 mg Instant Ocean salt per litre of distillate H2O), and we started feeding them with dry food (Gemma micro 75 μm, Skretting, I) twice per day. We kept the larvae in this condition until the start of the experiments. 2.2. Experiment 1 We followed the procedure typically used for fish and other groups to assess behavioural responses to alarm cues [37,38]. We tested 77 larvae at the age of 12 dpf and 76 larvae at the age of 24 dpf. Each subject was tested only once. On the day of the experiment, we individually moved each subject into a square well (4 × 4 cm) made of white acrylic plastic (Fig. 1a). The well was filled with 6 mL of water (60 mg Instant Ocean salt per litre of distillate H2O) kept at 28 ± 0.5 °C. Under the well, we placed two warm-white LED stripes that illuminated the apparatus uniformly from below because of the acrylic sheet’s opalescence. This setting prevented shadows and reflections on the water surface, which helped us to record the subject’s behaviour from above. We left the subjects undisturbed for 40 min before analysing their behaviour. In pilot studies, we found that the larvae exhibited increased activity for 30 min upon transportation in the wells. The 40-min acclimation time prevented the behavioural response to transportation from affecting the study results. After the acclimation, we recorded the subjects’ behaviour using a camera (Canon Legria HF R38) placed 1 m above the apparatus. Using a computer running the Ethovision software (v. 11.0, Noldus Information Technology, NL), we tracked the subject’s position for 12 min (Fig. 1b). This first observation period served to measure the baseline level of behaviour of each subject. Then, we administered the cues in the wells using a 2.5-ml syringe with a plastic needle. The needle was positioned close to the wall of the well and the cue was expelled gently to avoid disturbing the fish. We recorded the subject’s behaviour for another 12min observation period after exposure to the cue. Most studies on alarm cues in adult zebrafish and other species tested the subjects for 5−8 min (e.g., Ferrari et al. [39], Maximino et al. [40], Quadros et al. [41]). We used 12-min observation periods because we did not know whether the larvae would respond to the treatment as quickly as adults do. Each subject received 0.5 mL of one among three possible cues: alarm cues (12 dpf: N = 35; 24 dpf: N = 35), water control (12 dpf: N = 30; 24 dpf: N = 29), or fish odour control (12 dpf: N = 12; 24 dpf: N = 12). We described the subjects’ behaviour using three variables: distance moved (activity), frequency of entering the centre of the arena and time spent in the centre of the arena. The distance moved is the most common behavioural variable used to study response to alarm cue because fish markedly reduce their activity in the presence of predation risk [8–10]. We used the other two variables because they describe 2
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Fig. 1. (a) The system of wells used to test zebrafish larvae and detail of a larva in the well; light was provided by LED strips placed underneath the acrylic bottom of the well; this setting allows efficient locomotion tracking of the larvae using a camera placed on the ceiling. (b) Timeline of the procedure used in the three experiments.
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Council (18/2017-UT). The Ethical Committee of the University of Ferrara and the Italian Ministry of Health reviewed and approved the experimental procedures (protocol CB/2017 and 416/2018-PR).
thigmotaxis, a behaviour observed in zebrafish larvae that may be related to anxiety and thus response to alarm cue [42]. The centre of the arena was virtually delimited by a 3 × 3 cm squared area. Following previous studies on fish [29,43,31], we obtained alarm cue by euthanizing zebrafish larvae and homogenising their entire body with a surgical scalpel. We performed euthanasia with head concussion and did not use anaesthetic because doing so can interfere with the alarm cue [44]. Then, we suspended the larvae donors’ tissues in water at a concentration of 1 donor/mL. We used donors of the same age as the subjects. For the fish odour controls, we used the water in which we maintained two shark catfish (Pangasianodon hypophthalmus; 12 cm length; 4 L of water) for 24 h. To the best of our knowledge, shark catfish are not considered predators of zebrafish [45], and there are no reports of zebrafish innately responding to the cues of this species.
2.6. Statistical analysis We analysed the data in R (version 3.4.0; The R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org). In all of the experiments, the main dependent variable was the subjects’ activity, measured as distance moved (mm) in each minute of the testing phase (24 min overall; 12-min baseline observation period and 12-min of post-exposure observation period), which was log transformed before the analysis to deal with skewed distributions. In experiment 1, we also analysed the frequency of entering the centre of the arena (log transformation) and the proportion of time spent in the centre of the arena (arcsine square root transformation). We used generalised linear mixed-effects models fitted with the lmer function of the lme4 package. All of the models included subject ID as a random effect to account for repeated measurements. We calculated the effect significance using χ2 tests computed with the Anova function of the car package. In experiment 1, we initially fitted 2 × 3 × 2 models (one per dependent variable) with the following fixed effects: age (12 dpf versus 24 dpf), type of cue injected (alarm cue versus water control versus fish odour control), and observation period (baseline versus post-exposure). We then ran two separate models, one for each age group of fish. In experiments 2 and 3, we fitted a 2 × 2 model with the following fixed effects: conditioning cues (alarm cue plus fish odour versus water plus fish odour) and observation period (baseline versus post-exposure). For experiment 2, we also ran a tentative analysis including only the last minute of the baseline observation period, and the first minute of the post-exposure observation period to focus on the behavioural change and achieve more power in detecting an eventual effect. In experiment 1, we also used Pearson’s correlation tests to assess the relationship between the different dependent variables analysed.
2.3. Experiment 2 In this experiment, we used only 24-dpf larvae (N = 48) because they showed a clearer antipredator response to alarm cue than 12-dpf larvae did in experiment 1. We followed the typical procedure used to study alarm cue-mediated predator learning [39,46,47], which generally followed that of classical conditioning studies [48,49]. It consisted of two steps: the conditioning phase in which we presented alarm cue (unconditioned stimulus that produce an unconditioned response) paired with fish odour (conditioned stimulus that initially does not produce response) and the test phase in which we exposed the subjects to the fish odour alone to measure the conditioned response (Fig. 1b). We began the conditioning phase by moving the larvae into the wells for a 40-min acclimatisation. Then, we exposed one group of larvae (N = 24) to alarm cue paired with catfish odour, and the remaining half of the larvae (N = 24) were exposed to water plus catfish odour as control. We used catfish odour as the novel predator odour because the results of experiment 1 demonstrated that zebrafish do not respond to this species with innate antipredator behaviour. We injected 0.5 mL of each cue in the well, as described for the previous experiment. Cues were obtained as in experiment 1. We tested the larvae after 6 h by exposing them to 0.5 mL of catfish odour and tracking their locomotor activity, as described before. In this testing phase, we measured the presence of a learned response to the catfish cue. We recorded larvae’s activity during a baseline observation period (12 min) and in an observation period after exposure to the cue (12 min) as described above. We focused on activity because experiment 1 suggested that it was the most appropriate variable for detecting response to alarm cues in larvae. In case of learning, we expected a stronger decrease in activity between the baseline and the post-exposure observation period in the group conditioned with alarm cue.
3. Results 3.1. Experiment 1 3.1.1. Distance moved The model found significant main effects of type of cue (χ22 = 9.117, P = 0.010) and observation period (χ21 = 214.070, P < 0.0001), but not of larvae age (χ21 = 1.701, P = 0.192). Among the two-way interactions, the age × observation period and the type of cue × observation period were significant (χ21 = 35.432, P < 0.001 and χ22 = 134.738, P < 0.0001, respectively). The age × type of cue interaction was not significant (χ22 = 0.998, P = 0.607). The three-way interaction was significant (χ22 = 40.963, P < 0.0001), indicating that larvae responded differently to the different types of cue, and this response was modulated by larvae age. To clarify the role of age suggested by the three-way interaction, we run two additional models, one for each age group of larvae. In the model of the 12 dpf larvae, there was a significant effect of observation period (χ21 = 29.602, P < 0.0001), but no significant effect of type of cue (χ22 = 4.301, P = 0.116). The interaction between these two terms was significant (χ22 = 25.538, P < 0.0001). This indicated that 12 dpf larvae responded to the cue with a change in activity. In particular, there was a larger decrease in activity in response to alarm cue compared to the exposure to the two control cues, water and catfish odour (Fig. 2a). In the model of the 24 dpf larvae, there was a significant effect of observation period (χ21 = 295.695, P < 0.0001) and type of cue (χ22 = 7.653, P = 0.022). The interaction between these two terms was significant (χ22 = 199.388, P < 0.0001). This indicated that 24 dpf larvae also responded to the cue with a change in activity: there was a larger decrease in activity in response to alarm cue compared to the
2.4. Experiment 3 Experiment 3 was identical to the experiment 2 with one exception. To increase the interval between the conditioning and testing phases, 40 min after conditioning, we moved the larvae into maintenance tanks divided by experimental treatment (alarm cue conditioning and water control). We then tested the larvae after 24 h (Fig. 1b). In total, we used 36 larvae at 24 dpf. The testing phase was performed as described for experiment 2, after a 40-min habituation period to the wells. We tested two groups of larvae; 18 larvae exposed to alarm cues plus catfish odour and 18 larvae exposed to catfish odour plus water as control. 2.5. Ethical note All of the husbandry and experimental procedures were performed in accordance with the European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU) and the Italian animal-protection standards (D.lgs. 26/2014). We obtained general license for fish maintenance and breeding for the Ferrara City 4
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Fig. 2. Activity (distance moved) of (a) 12-dpf and (b) 24-dpf zebrafish in each minute of experiment 1. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue (either alarm cue, water control, or fish odour control; see colour legend).
exposure to the two control cues, water and catfish odour (Fig. 2b).
a clear response to alarm cue (Fig. 4a and b).
3.1.2. Frequency of entering the centre of the arena The analysis on the frequency of entering the centre of the arena provided results similar to that of distance moved. The initial model revealed a significant three-way interaction (χ22 = 24.118, P < 0.0001; Fig. 3a and b). There were significant main effects (type of cue: χ22 = 32.715, P < 0.0001; observation period: χ21 = 316.834, P < 0.0001; and larvae age: χ21 = 12.997, P < 0.0001) and significant two-way interactions (age × observation period: χ21 = 61.493, P < 0.001; type of cue × observation period: χ22 = 122.960, P < 0.0001; age × type of cue: χ22 = 13.073, P < 0.001). The models analysing subjects of the two ages separately revealed that both 12- and 24-dpf larvae showed a different frequency of entering the centre of the arena according to the type of cue (12 dpf: χ22 = 29.513, P < 0.0001; 24 dpf: χ22 = 120.981, P < 0.0001). For both ages, alarm cue seems to cause a decrease in activity more marked compared to the other cues (Fig. 3a and b). However, results were less clear compared to those of the distance moved. Models also found significant main effects (12 dpf, type of cue: χ22 = 26.969, P < 0.0001; 12 dpf, observation period: χ21 = 47.880, P < 0.0001; 24 dpf, type of cue: χ22 = 16.959, P < 0.0001; 24 dpf, observation period: χ21 = 342.585, P < 0.0001).
3.1.4. Correlation between different behavioural measures The proportion of time spent in the centre of arena was positively correlated with the distance moved (r151 = 0.638, P < 0.0001), but there was no correlation between the frequency of entering the centre of the arena and the distance moved (r151 = -0.076, P = 0.350). 3.2. Experiment 2 The model revealed a significant effect of observation period (χ21 = 71.619, P < 0.0001), indicating that larvae decreased their activity from the baseline to the post-exposure observation period (Fig. 4a). The effect of conditioning cues was also significant (χ21 = 5.815, P = 0.016): larvae conditioned with alarm cue plus catfish odour showed on average reduced activity compared to larvae exposed to control water plus catfish odour during the conditioning phase (Fig. 4a). The observation period × conditioning cues interaction was not significant (χ21 = 0.026, P = 0.872), which provided no support for the fact that larvae learned to recognise the novel odour via pairing with alarm cue. When we restricted the analysis on the last minute of the baseline observation period and the first minute of the post-exposure observation period, we found a significant observation period × conditioning cues interaction (χ21 = 6.071, P = 0.014; main effect of observation period: χ21 = 20.053, P < 0.0001; main effect of conditioning cues: χ21 = 6.904, P = 0.009).
3.1.3. Proportion of time spent in the centre of the arena The analysis on the proportion of time spent in the centre of the arena provided different results compared to previous analyses. The three main effects in the initial model were significant (type of cue: χ22 = 11.182, P = 0.004; observation period: χ21 = 4.183, P = 0.041; and larvae age: χ21 = 28.292, P < 0.0001) and two of the two-way interactions were significant (age × observation period: χ21 = 0.250, P = 0.617; type of cue × observation period: χ22 = 18.172, P = 0.0001; age × type of cue: χ22 = 6.452, P = 0.040). However, the three-way interaction was not significant (χ22 = 5.781, P = 0.556; Fig. 4a and b). For this dependent variable, it was not possible to detect
3.3. Experiment 3 The model revealed a significant effect of observation period (χ21 = 119.338, P < 0.0001), but not significant effect of conditioning cues (χ21 = 0.001, P = 0.992). Larvae conditioned with alarm cue plus catfish odour showed on average similar activity compared to larvae exposed to control water plus catfish odour during the conditioning 5
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Fig. 3. Frequency of entering the centre of the arena of (a) 12-dpf and (b) 24-dpf zebrafish in each minute of experiment 1 and proportion of time spent in the centre of the arena (arcsine square root transformation) of (c) 12-dpf and (d) 24-dpf zebrafish in each minute of experiment 1. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue (either alarm cue, water control, or fish odour control; see colour legend).
pairing with alarm cue.
phase (Fig. 4b). The observation period × conditioning cues interaction was significant (χ21 = 51.485, P < 0.0001). Larvae conditioned with alarm cue showed a greater reduction in activity in the post-exposure observation period compared to larvae exposed to water during conditioning, as expected if larvae learned to recognise the novel odour via
4. Discussion Behavioural responses induced by alarm cues may notably favour 6
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Fig. 4. Activity (distance moved) of zebrafish in each minute of the testing phase of (a) experiment 2 and (b) experiment 3. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue in the testing phase (catfish odour).
characterising the ontogeny of zebrafish alarm cue at the chemical level. However, this is a difficult task because the composition of alarm cue is mostly unknown and probably consists of a mixture of many substances, with only some of them having been characterised yet (blood: [17]; chondroitin: [16]; hypoxanthine 3-N-oxide [14]:). For researchers interested in using alarm cue response as a model, our results suggest to test zebrafish aged approximately 24 dpf and to avoid the use of younger subjects, which can show a weaker response. The results of experiment 1 indicate that zebrafish larvae are sensitive to the alarm cue of conspecifics of the same age, which can pave the way to developing models in several fields of translational research. Future studies should try to understand the mechanisms causing the alarm cue-induced behavioural responses and the effects of the exposure to alarm cue in zebrafish larvae. The first point to be addressed is the identity of the molecules perceived as alarm cue by larvae. Indeed, we obtained alarm cue by homogenising the entire donor, as commonly due to study small species and early developmental stages [29,54]. Literature suggests hypoxanthine-3 N-oxide [12–14] and glycosaminoglycan chondroitin [16] as candidate molecules, although most authors recognise alarm cue to be a mixture of chemicals (reviewed in Chivers et al. [20]). The second relevant point to address is which neural partway controls alarm cue response. In adult zebrafish, the response to alarm cue has been mainly characterised using pharmacological methods. The serotonergic system seems to be involved because the brain of adult zebrafish exposed to alarm cue shows increased extracellular 5-HT levels [40]. Cholinergic signalling is also involved because nicotine blocks the effects of alarm cue in adult zebrafish [55]. In adult zebrafish, alarm cue also causes an increase in blood haemoglobin, glucose, epinephrine and norepinephrine levels [40]. Some studies tried to identify the neural substrates responsible for reaction to alarm cue. In the crucian carp, Carassius carassius, surgical ablation experiments have revealed that alarm cue reaction is mediated by sensory neurons making synaptic connections with the medial bundle of the medial olfactory tract [56]. Mathuru and colleagues [16] reported that in adult and 28–50 dpf zebrafish, skin extracts activated
research on zebrafish anxiety, fear, stress and cognitive functions such as learning. This contribution might also be relevant for research on larvae, but their behavioural response to alarm cues has not yet been documented. Here, we provided evidence that zebrafish larvae exhibit innate response to alarm cues obtained by homogenising conspecific larvae. In addition, zebrafish larvae could be conditioned by pairing these alarm cues with a novel odour. In experiment 1, we exposed 12- and 24-dpf zebrafish to conspecific alarm cue to study their innate behavioural response. We found reduced activity among the larvae exposed to the alarm cue, stronger than that due to the experimental manipulation and the exposure to a novel fish cue, as suggested by the comparison with the control groups. Similar conclusions were reached considering one measure of thigmotaxis—the frequency of entering the centre of the arena—though the effects were substantially less clear than those obtained from activity. The second measure of thigmotaxis, the proportion of time spent in the centre of the arena, was correlated with the activity but did not evidenced the alarm cue’s effect. Therefore, it seems reasonable that the activity is more appropriate for studying alarm cue responses in larvae. The analyses on activity separated per age suggested that the larvae responded to alarm cue at both 12 and 24 dpf. However, the response to the alarm cue was much stronger in 24-dpf compared to 12-dpf zebrafish, as indicated by the significant three-way interaction and by plotting (Fig. 2). There are at least two possible explanations for the age difference. First, larvae in the younger group might not have fully developed the nervous substrates necessary to detect and recognise alarm cue, which might cause a weaker response. The number of neurons in the zebrafish olfactory bulb after hatching is lower compared to that of adults (∼5 %) and subsequently increases with ontogeny [50,51]. On the other hand, the alarm cue of young larvae may be produced in smaller quantities, and the response to alarm cue is often dose dependent [52] and/or the alarm cue of young larvae is less efficient in evoking the response. For example, male fathead minnows, Pimephales promelas, do not produce active alarm substances during the breeding season [53]. The age difference might be further investigated by
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based on other forms of associative learning usually require extensive and time-consuming trainings [60]. On the other hand, some characteristics of predator recognition learning might represent confounding factors and should be carefully considered. In contrast with other forms of learning, multiple conditioning events with alarm cue might be detrimental. When risk persists for a considerable amount of time, prey often dramatically reduce responses to the predator, to counterbalance the loss of resources due to antipredator behaviour, such as hiding, which occurs during the initial exposure to the risk (risk allocation: [61]). Similarly, it is difficult to assess memory window for predator recognition learning, although this theoretically requires little change in the procedure that we adopted to study learning. Indeed, prey might stop responding to a predator cue even if they still recognise it as dangerous. Another possible limitation regards discrimination learning because prey tend to respond to olfactory cues somehow similar to that used during conditioning (generalization of predator recognition [46]). In our study, we used a wild-type outbreed strain of zebrafish, whereas most of the translational research exploits inbreed and genetically modified lines. It should be verified whether in other zebrafish strains, larvae’s response to alarm cues is similar to that observed in this study. However, we do not expect large differences between strains. The main variable measured in this study, activity reduction, is highly reliable and showed by almost all fish species investigated (e.g., Ferrari et al. [10]). Among adults, different strains respond differently to alarm cues [41,62], but these differences were usually limited to behavioural measures other than activity reduction (e.g., number of vertical drifts, erratic movements, and in angular velocity). In conclusion, our study revealed that zebrafish larvae exhibit direct behavioural responses to alarm cue and use it to develop a learned antipredator response. Recent studies with a similar experimental approach but performed with adult zebrafish are unravelling how alarm cue responses vary by the context, the paradigm and the population of fish used and how alarm cue modulates aggression, the monoaminergic system and spatial behaviour [62–64]. It will be interesting to investigate these factors in larvae to further evaluate the possibility of developing behavioural and cognitive models based on zebrafish at the larval stage. These models might benefit from being quick to perform and exploiting the zebrafish’s natural antipredator behaviour.
the mediodorsal posterior olfactory bulb, which has a projection to the habenula. A similar activation was reported for 3-week-old zebrafish, despite the lack of a behavioural response to the substance. These data suggest that the response of larvae can be mediated by the same neural substrates as for adults. The c-fos expression analyses and the wholebrain imaging techniques available for freely swimming larvae [7,57] might provide important advancements for understanding the neurons involved in alarm cue response. Experiment 2 was aimed at studying whether 24-dpf zebrafish could be conditioned to recognise a novel odour as dangerous via pairing with alarm cue. In case of learning, larvae were expected to show an antipredator response (activity reduction) to the conditioned odour administered in testing phase. When considering the entire testing time, we found no evidence of a learned antipredator response in larvae conditioned with alarm cue plus a novel fish odour: their response was not detectably different from that of control larvae exposed to water during the conditioning phase. Larvae might not be able learn to recognise a novel odour as dangerous via pairing with alarm cue, even if experiment 1 provided evidence that they can perceive the alarm cue. A similar ontogenetic effect on cognitive abilities has been reported for another small teleost fish, the guppy, Poecilia reticulata. Individuals of this species do not reach their maximum accuracy in a cognitive task until the age of 40 days [58]. However, an analysis restricted to the minute before and after cue administration revealed a slightly stronger response to the novel fish odour, in terms of activity reduction, in larvae conditioned with alarm cue, as compared to controls exposed to water during the conditioning phase. This effect leads to a second, methodological explanation. We might have failed to detect the learned antipredator response because this effect was masked by other factors. Larvae conditioned with alarm cue had much lower activity in the test phase, even before the exposure to testing cues (Fig. 4a), as compared to larvae exposed to water during conditioning. This lower activity was probably a mechanism of dealing with the risk of predation and went on for up to 6 h after the conditioning phase (i.e., when the test phase took place). To verify this interpretation, we ran experiment 3 using a greater interval between the conditioning and the test phase (24 h). In experiment 3, we did not observe reduced baseline activity among larvae exposed to alarm cue (Fig. 4b). Also, the effect of the learned antipredator response was detected. Therefore, experiments 2 and 3 provided evidence that zebrafish larvae at the age of 24 dpf can learn to recognise a novel predator cue when it is paired with alarm cue, but that this effect is clearly visible only with an extended interval between conditioning and testing. To date, paradigms for studying cognition in zebrafish larvae are mostly based on simple forms of learning, such as habituation and sensitisation [59]. Alarm cue-based learning might allow developing paradigms to study more complex cognitive abilities, such as associative learning. Beside training larvae to recognise a novel odour, it might be possible to train them to associate danger with a specific visual cue and to study visual discrimination learning. For example, larvae can be exposed to two objects, including one associated with alarm cue, allowing researchers to test whether larvae can distinguish between the two objects. It may also be possible to use alarm cue to study spatial learning. Brown (2003) [8,9] presented rainbowfish, Melanotaenia spp., with a predator model placed in a particular microhabitat, and the fish learned to avoid the ‘dangerous’ microhabitat. A similar learning process might be triggered by alarm cue instead of a predator model and be used to investigate spatial abilities in larval zebrafish. It may be also possible to assess mnemonic retention because the association between a cue and the danger usually lasts for many days [48]. Using these and similar paradigms might allow for assessing learning differences between larvae with different genotypes or exposed to different pharmacological treatments, with two great advantages. First, complex cognitive processes can be studied by measuring a rather simple behavioural response (activity reduction). Second, experiments with these paradigms would require only one training session, whereas paradigms
CRediT authorship contribution statement Tyrone Lucon-Xiccato: Conceptualization, Methodology, Formal analysis, Funding acquisition, Writing - original draft. Giuseppe Di Mauro: Conceptualization, Data curation, Writing - review & editing. Angelo Bisazza: Conceptualization, Funding acquisition, Writing - review & editing. Cristiano Bertolucci: Conceptualization, Funding acquisition, Writing - review & editing. Acknowledgements We have no competing interests. This work was supported by FAR2018, FAR2019 and FIR2018 from University of Ferrara to TLX and CB, by DOR grant from Università di Padova to AB, and by 31 st PhD program in Evolutionary Biology and Ecology at the University of Ferrara. References [1] R.J. Egan, C.L. Bergner, P.C. Hart, J.M. Cachat, P.R. Canavello, M.F. Elegante, et al., Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish, Behav. Brain Res. 205 (2009) 38–44. [2] S. Jesuthasan, Fear, anxiety, and control in the zebrafish, Dev. Neurobiol. 72 (2012) 395–403. [3] B.V. Hope, T.J. Hamilton, P.L. Hurd, Submerged plus maze: a novel test for studying anxiety-like behaviour in fish, Behav. Brain Res. 362 (2019) 332–337. [4] C. Maximino, T.M. de Brito, A.W. da Silva Batista, A.M. Herculano, S. Morato,
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Research report
Alarm cue-mediated response and learning in zebrafish larvae a,
a
b
Tyrone Lucon-Xiccato *, Giuseppe Di Mauro , Angelo Bisazza , Cristiano Bertolucci a b
T a
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Dipartimento di Psicologia Generale, Università di Padova, Padova, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Alarm substances Fish behaviour Fish cognition Predator recognition Learning
We investigated the behavioural and learning response of zebrafish larvae to chemicals released by injured conspecifics (the alarm cue). Many aquatic vertebrates and invertebrates exhibit an innate antipredator response to alarm cues because in nature, they reliably indicate the presence of predators. Likewise, when an individual simultaneously perceives a novel odour and alarm cue, it learns to recognise the novel odour as a predator odour. Alarm cue-mediated behavioural response and learning have been reported in some fish and amphibians during early ontogeny, but in zebrafish, they have been described only for adults. In this study, we demonstrated that zebrafish at 12 and 24 days post fertilization exhibited reduced activity when exposed to alarm cue obtained by homogenised larvae of the same age, with this response being greater for the older zebrafish. In addition, we showed that 24-dpf zebrafish conditioned with alarm cue plus a novel odour learned to recognise the novel odour as a threat and responded to it with antipredator behaviour. The innate behavioural response and the learned response after conditioning may be used to develop paradigms with which to study anxiety, fear, stress, learning and memory in zebrafish larvae.
1. Introduction
species revealed that even their ink evokes alarm response [19]. Alarm cues are considered by most authors a mixture of different substances released upon damage that fish and other aquatic organisms use both individually, and collectively to identify predation risk (reviewed in Chivers et al. [20]). The zebrafish is also increasingly used for research on learning and memory [21–23], and alarm cues might be useful in this regard. Indeed, adult zebrafish, and other aquatic organisms, exploit alarm cue to learn about novel predators [24,25]. When a prey perceives alarm cue paired with a novel odour, it learns to recognise the novel odour as a threat. The conditioned response consists of antipredator behaviour (e.g., decreased activity) when exposed again to the conditioned cue (i.e., the novel odour), even without alarm cue [8–10]. This association between alarm cue and novel odour is extremely robust and occurs at the first presentation of the stimuli [10]. Intuitively, selection has favoured evolution of this one-trial learning mechanism to allow predator recognition after a single exposure, thereby increasing the prey’s chances of survival in future encounters [10]. Translational research is often interested in the use of larvae (i.e., age < 30 days post fertilisation, dpf) rather than of adult zebrafish [26]. Larvae can be collected in large numbers and tested early, given their quick development, making them particularly suitable to largescale screenings of drugs and genotypes. The innate behavioural
The zebrafish is gaining increasing importance as model for fear, stress and anxiety disorders [1,2]. For this reason, several behavioural paradigms have been developed (e.g. Hope et al. [3], Maximino et al. [4,5]), some of which exploit the innate response to alarm cues [6,7]. Alarm cues are substances contained in the skin and other tissues of many aquatic species [8–10]. When a prey is captured by a predator, the former’s damaged tissues release alarm cue in the water, which conspecifics can perceive via the olfactory system and use as a signal of danger to perform antipredator behaviours [8–10]. Adult zebrafish respond to alarm cues with typical antipredator behaviours, such as hiding or freezing, which cause a marked decrease in the locomotor activity [7]. Despite alarm cues having being studied for almost one century [11], their exact composition is unresolved. A line of evidence has suggested that hypoxanthine-3 N-oxide is the main substance causing antipredator response in fish [12,13], including in zebrafish [14]. However, this substance was not reliably detected in fish skin [15]. A recent biochemical fractionation study on zebrafish identified glycosaminoglycan chondroitin as the active substance [16]. Barreto and colleagues [17] reported that conspecific blood also evokes alarm response in Nile Tilapia. The alarm-cue effect of blood was detected also by studies on crustaceans [18], whereas a study on a cephalopod
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Corresponding author at: Department of Life Sciences and Biotechnology, Via L. Borsari 46, 44121, Ferrara, Italy. E-mail address: [email protected] (T. Lucon-Xiccato).
https://doi.org/10.1016/j.bbr.2019.112446 Received 2 July 2019; Received in revised form 18 December 2019; Accepted 19 December 2019 Available online 20 December 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Brain Research 380 (2020) 112446
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2. Materials and methods
response to alarm cue and the learned response to stimuli conditioned with alarm cue could be ideal for studying anxiety, fear and stress responses, as well as learning and memory, in zebrafish larvae. However, to the best of our knowledge, larvae’s behavioural responses to alarm cue have not been used as a research model. A possible explanation is that an early study has reported the onset of zebrafish’s alarm cue response at the age of 48–52 dpf [27]. One may argue that this result seems somehow counterintuitive. Being able to respond to and recognise predators should substantially increase the chance of survival for zebrafish larvae, as reported in other species [28]. Therefore, there should be strong selection for early development of this ability. In line with this idea, response to alarm cues has been consistently documented in juvenile fish and amphibians, even at the embryonic stage [29–34]. For example, Atherton and McCormick [29] monitored the heart beating of embryonic cinnamon clownfish, Amphiprion melanopus, exposed to conspecific alarm cue, finding significant increase in beating rates. This result was interpreted as evidence that cinnamon clownfish embryos can recognise alarm cue. Detecting zebrafish larvae’s responses to alarm cue may be difficult because of methodological issues. Larvae might respond differently or more subtly to alarm cues compared to adults; therefore, most of the methodologies used for adults might not be adequate for larvae. Another potential confound is that, in the early study, zebrafish larvae were exposed to the alarm cues of older conspecifics [27]. In two fish species—brook char, Salvelinus fontinalis, and spiny chromis, Acanthochromis polyacanthus—individuals can distinguish the age of alarm cue donors and preferentially respond to the alarm cue of individuals of the same age [35,36]. Because size increases with age in most of fish species, alarm cue from larger or smaller conspecifics may indicate the presence of a predator that is not dangerous because it preys on individuals of a different age class. It is therefore possible that zebrafish larvae do not respond to the alarm cue of older conspecifics because it indicates the presence of a predator species that preys on older conspecifics and does not represent an immediate threat. In experiment 1, we tested whether zebrafish larvae respond to alarm cue from same-age conspecifics. To detect the larvae’s response, we measured activity reduction and thigmotaxis using an automatic high-throughput tracking system, which was expected to improve the detection of the larvae’s behavioural responses. We compared three groups of larvae: one group exposed to alarm cue; one group exposed to water as a control, to ensure that the response in the alarm cue-exposed group was not due to the experimental manipulation; and one group exposed to fish odour as a further control for responses due to general fish odours or novel odours. We performed this experiment using subjects at 12 and 24 dpf. In experiment 2, we investigated whether larvae can use alarm cue to learn to recognise a novel odour as a threat. First, we conditioned 24dpf zebrafish using alarm cue paired with the odour of a novel, unfamiliar fish species to simulate a predator. After 6 h, we tested the larvae’s conditioned response to the fish odour alone; we also observed the response of a group of larvae exposed to water plus fish odour as control. Following a commonly-used approach [10], we assessed learning exploiting the antipredator response of larvae, which was identified in experiment 1 (i.e., activity reduction). In case of alarm cuemediated learning, we expected that the group conditioned with alarm cues and fish odour would thereafter show a significantly stronger decrease in activity when exposed to fish odour, as compared to the control group pseudoconditioned with water. Because we suspected that a carryover effect had occurred in experiment 2, causing fish exposed to alarm cues to show reduced baseline activity, we performed experiment 3 with a larger interval (24 h) between the conditioning phase and the test phase.
2.1. Animal husbandry We performed our experiments using larvae of wild-type zebrafish. The adults used for reproduction were maintained according to standard protocols in our laboratory at the University of Ferrara. Briefly, maintenance 200-L glass tanks housed mixed sex-groups with approximately 40 individuals. A heater kept the water temperature at 28 ± 1 °C, and illumination was provided with a 14:10 LD cycle. We fed the animals live brine shrimp nauplii (Ocean Nutrition, USA) and commercial flakes (Sera GmbH, D) twice per day. For reproduction, we transferred pairs of adult zebrafish into a breeding cage (Techniplast, I) in the late afternoon. We kept the two breeders divided by a transparent partition until light on of the following day, after which we removed the partition and the fish could spawn. We immediately collected the embryos and raised them in a Petri dish filled with E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSO4; 1 % of methylene blue). When they reached the age of 5 dpf, we moved the larvae into small glass tanks (20 × 15 cm, height 10 cm) filled with 2 cm of water (60 mg Instant Ocean salt per litre of distillate H2O), and we started feeding them with dry food (Gemma micro 75 μm, Skretting, I) twice per day. We kept the larvae in this condition until the start of the experiments. 2.2. Experiment 1 We followed the procedure typically used for fish and other groups to assess behavioural responses to alarm cues [37,38]. We tested 77 larvae at the age of 12 dpf and 76 larvae at the age of 24 dpf. Each subject was tested only once. On the day of the experiment, we individually moved each subject into a square well (4 × 4 cm) made of white acrylic plastic (Fig. 1a). The well was filled with 6 mL of water (60 mg Instant Ocean salt per litre of distillate H2O) kept at 28 ± 0.5 °C. Under the well, we placed two warm-white LED stripes that illuminated the apparatus uniformly from below because of the acrylic sheet’s opalescence. This setting prevented shadows and reflections on the water surface, which helped us to record the subject’s behaviour from above. We left the subjects undisturbed for 40 min before analysing their behaviour. In pilot studies, we found that the larvae exhibited increased activity for 30 min upon transportation in the wells. The 40-min acclimation time prevented the behavioural response to transportation from affecting the study results. After the acclimation, we recorded the subjects’ behaviour using a camera (Canon Legria HF R38) placed 1 m above the apparatus. Using a computer running the Ethovision software (v. 11.0, Noldus Information Technology, NL), we tracked the subject’s position for 12 min (Fig. 1b). This first observation period served to measure the baseline level of behaviour of each subject. Then, we administered the cues in the wells using a 2.5-ml syringe with a plastic needle. The needle was positioned close to the wall of the well and the cue was expelled gently to avoid disturbing the fish. We recorded the subject’s behaviour for another 12min observation period after exposure to the cue. Most studies on alarm cues in adult zebrafish and other species tested the subjects for 5−8 min (e.g., Ferrari et al. [39], Maximino et al. [40], Quadros et al. [41]). We used 12-min observation periods because we did not know whether the larvae would respond to the treatment as quickly as adults do. Each subject received 0.5 mL of one among three possible cues: alarm cues (12 dpf: N = 35; 24 dpf: N = 35), water control (12 dpf: N = 30; 24 dpf: N = 29), or fish odour control (12 dpf: N = 12; 24 dpf: N = 12). We described the subjects’ behaviour using three variables: distance moved (activity), frequency of entering the centre of the arena and time spent in the centre of the arena. The distance moved is the most common behavioural variable used to study response to alarm cue because fish markedly reduce their activity in the presence of predation risk [8–10]. We used the other two variables because they describe 2
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Fig. 1. (a) The system of wells used to test zebrafish larvae and detail of a larva in the well; light was provided by LED strips placed underneath the acrylic bottom of the well; this setting allows efficient locomotion tracking of the larvae using a camera placed on the ceiling. (b) Timeline of the procedure used in the three experiments.
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Council (18/2017-UT). The Ethical Committee of the University of Ferrara and the Italian Ministry of Health reviewed and approved the experimental procedures (protocol CB/2017 and 416/2018-PR).
thigmotaxis, a behaviour observed in zebrafish larvae that may be related to anxiety and thus response to alarm cue [42]. The centre of the arena was virtually delimited by a 3 × 3 cm squared area. Following previous studies on fish [29,43,31], we obtained alarm cue by euthanizing zebrafish larvae and homogenising their entire body with a surgical scalpel. We performed euthanasia with head concussion and did not use anaesthetic because doing so can interfere with the alarm cue [44]. Then, we suspended the larvae donors’ tissues in water at a concentration of 1 donor/mL. We used donors of the same age as the subjects. For the fish odour controls, we used the water in which we maintained two shark catfish (Pangasianodon hypophthalmus; 12 cm length; 4 L of water) for 24 h. To the best of our knowledge, shark catfish are not considered predators of zebrafish [45], and there are no reports of zebrafish innately responding to the cues of this species.
2.6. Statistical analysis We analysed the data in R (version 3.4.0; The R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org). In all of the experiments, the main dependent variable was the subjects’ activity, measured as distance moved (mm) in each minute of the testing phase (24 min overall; 12-min baseline observation period and 12-min of post-exposure observation period), which was log transformed before the analysis to deal with skewed distributions. In experiment 1, we also analysed the frequency of entering the centre of the arena (log transformation) and the proportion of time spent in the centre of the arena (arcsine square root transformation). We used generalised linear mixed-effects models fitted with the lmer function of the lme4 package. All of the models included subject ID as a random effect to account for repeated measurements. We calculated the effect significance using χ2 tests computed with the Anova function of the car package. In experiment 1, we initially fitted 2 × 3 × 2 models (one per dependent variable) with the following fixed effects: age (12 dpf versus 24 dpf), type of cue injected (alarm cue versus water control versus fish odour control), and observation period (baseline versus post-exposure). We then ran two separate models, one for each age group of fish. In experiments 2 and 3, we fitted a 2 × 2 model with the following fixed effects: conditioning cues (alarm cue plus fish odour versus water plus fish odour) and observation period (baseline versus post-exposure). For experiment 2, we also ran a tentative analysis including only the last minute of the baseline observation period, and the first minute of the post-exposure observation period to focus on the behavioural change and achieve more power in detecting an eventual effect. In experiment 1, we also used Pearson’s correlation tests to assess the relationship between the different dependent variables analysed.
2.3. Experiment 2 In this experiment, we used only 24-dpf larvae (N = 48) because they showed a clearer antipredator response to alarm cue than 12-dpf larvae did in experiment 1. We followed the typical procedure used to study alarm cue-mediated predator learning [39,46,47], which generally followed that of classical conditioning studies [48,49]. It consisted of two steps: the conditioning phase in which we presented alarm cue (unconditioned stimulus that produce an unconditioned response) paired with fish odour (conditioned stimulus that initially does not produce response) and the test phase in which we exposed the subjects to the fish odour alone to measure the conditioned response (Fig. 1b). We began the conditioning phase by moving the larvae into the wells for a 40-min acclimatisation. Then, we exposed one group of larvae (N = 24) to alarm cue paired with catfish odour, and the remaining half of the larvae (N = 24) were exposed to water plus catfish odour as control. We used catfish odour as the novel predator odour because the results of experiment 1 demonstrated that zebrafish do not respond to this species with innate antipredator behaviour. We injected 0.5 mL of each cue in the well, as described for the previous experiment. Cues were obtained as in experiment 1. We tested the larvae after 6 h by exposing them to 0.5 mL of catfish odour and tracking their locomotor activity, as described before. In this testing phase, we measured the presence of a learned response to the catfish cue. We recorded larvae’s activity during a baseline observation period (12 min) and in an observation period after exposure to the cue (12 min) as described above. We focused on activity because experiment 1 suggested that it was the most appropriate variable for detecting response to alarm cues in larvae. In case of learning, we expected a stronger decrease in activity between the baseline and the post-exposure observation period in the group conditioned with alarm cue.
3. Results 3.1. Experiment 1 3.1.1. Distance moved The model found significant main effects of type of cue (χ22 = 9.117, P = 0.010) and observation period (χ21 = 214.070, P < 0.0001), but not of larvae age (χ21 = 1.701, P = 0.192). Among the two-way interactions, the age × observation period and the type of cue × observation period were significant (χ21 = 35.432, P < 0.001 and χ22 = 134.738, P < 0.0001, respectively). The age × type of cue interaction was not significant (χ22 = 0.998, P = 0.607). The three-way interaction was significant (χ22 = 40.963, P < 0.0001), indicating that larvae responded differently to the different types of cue, and this response was modulated by larvae age. To clarify the role of age suggested by the three-way interaction, we run two additional models, one for each age group of larvae. In the model of the 12 dpf larvae, there was a significant effect of observation period (χ21 = 29.602, P < 0.0001), but no significant effect of type of cue (χ22 = 4.301, P = 0.116). The interaction between these two terms was significant (χ22 = 25.538, P < 0.0001). This indicated that 12 dpf larvae responded to the cue with a change in activity. In particular, there was a larger decrease in activity in response to alarm cue compared to the exposure to the two control cues, water and catfish odour (Fig. 2a). In the model of the 24 dpf larvae, there was a significant effect of observation period (χ21 = 295.695, P < 0.0001) and type of cue (χ22 = 7.653, P = 0.022). The interaction between these two terms was significant (χ22 = 199.388, P < 0.0001). This indicated that 24 dpf larvae also responded to the cue with a change in activity: there was a larger decrease in activity in response to alarm cue compared to the
2.4. Experiment 3 Experiment 3 was identical to the experiment 2 with one exception. To increase the interval between the conditioning and testing phases, 40 min after conditioning, we moved the larvae into maintenance tanks divided by experimental treatment (alarm cue conditioning and water control). We then tested the larvae after 24 h (Fig. 1b). In total, we used 36 larvae at 24 dpf. The testing phase was performed as described for experiment 2, after a 40-min habituation period to the wells. We tested two groups of larvae; 18 larvae exposed to alarm cues plus catfish odour and 18 larvae exposed to catfish odour plus water as control. 2.5. Ethical note All of the husbandry and experimental procedures were performed in accordance with the European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU) and the Italian animal-protection standards (D.lgs. 26/2014). We obtained general license for fish maintenance and breeding for the Ferrara City 4
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Fig. 2. Activity (distance moved) of (a) 12-dpf and (b) 24-dpf zebrafish in each minute of experiment 1. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue (either alarm cue, water control, or fish odour control; see colour legend).
exposure to the two control cues, water and catfish odour (Fig. 2b).
a clear response to alarm cue (Fig. 4a and b).
3.1.2. Frequency of entering the centre of the arena The analysis on the frequency of entering the centre of the arena provided results similar to that of distance moved. The initial model revealed a significant three-way interaction (χ22 = 24.118, P < 0.0001; Fig. 3a and b). There were significant main effects (type of cue: χ22 = 32.715, P < 0.0001; observation period: χ21 = 316.834, P < 0.0001; and larvae age: χ21 = 12.997, P < 0.0001) and significant two-way interactions (age × observation period: χ21 = 61.493, P < 0.001; type of cue × observation period: χ22 = 122.960, P < 0.0001; age × type of cue: χ22 = 13.073, P < 0.001). The models analysing subjects of the two ages separately revealed that both 12- and 24-dpf larvae showed a different frequency of entering the centre of the arena according to the type of cue (12 dpf: χ22 = 29.513, P < 0.0001; 24 dpf: χ22 = 120.981, P < 0.0001). For both ages, alarm cue seems to cause a decrease in activity more marked compared to the other cues (Fig. 3a and b). However, results were less clear compared to those of the distance moved. Models also found significant main effects (12 dpf, type of cue: χ22 = 26.969, P < 0.0001; 12 dpf, observation period: χ21 = 47.880, P < 0.0001; 24 dpf, type of cue: χ22 = 16.959, P < 0.0001; 24 dpf, observation period: χ21 = 342.585, P < 0.0001).
3.1.4. Correlation between different behavioural measures The proportion of time spent in the centre of arena was positively correlated with the distance moved (r151 = 0.638, P < 0.0001), but there was no correlation between the frequency of entering the centre of the arena and the distance moved (r151 = -0.076, P = 0.350). 3.2. Experiment 2 The model revealed a significant effect of observation period (χ21 = 71.619, P < 0.0001), indicating that larvae decreased their activity from the baseline to the post-exposure observation period (Fig. 4a). The effect of conditioning cues was also significant (χ21 = 5.815, P = 0.016): larvae conditioned with alarm cue plus catfish odour showed on average reduced activity compared to larvae exposed to control water plus catfish odour during the conditioning phase (Fig. 4a). The observation period × conditioning cues interaction was not significant (χ21 = 0.026, P = 0.872), which provided no support for the fact that larvae learned to recognise the novel odour via pairing with alarm cue. When we restricted the analysis on the last minute of the baseline observation period and the first minute of the post-exposure observation period, we found a significant observation period × conditioning cues interaction (χ21 = 6.071, P = 0.014; main effect of observation period: χ21 = 20.053, P < 0.0001; main effect of conditioning cues: χ21 = 6.904, P = 0.009).
3.1.3. Proportion of time spent in the centre of the arena The analysis on the proportion of time spent in the centre of the arena provided different results compared to previous analyses. The three main effects in the initial model were significant (type of cue: χ22 = 11.182, P = 0.004; observation period: χ21 = 4.183, P = 0.041; and larvae age: χ21 = 28.292, P < 0.0001) and two of the two-way interactions were significant (age × observation period: χ21 = 0.250, P = 0.617; type of cue × observation period: χ22 = 18.172, P = 0.0001; age × type of cue: χ22 = 6.452, P = 0.040). However, the three-way interaction was not significant (χ22 = 5.781, P = 0.556; Fig. 4a and b). For this dependent variable, it was not possible to detect
3.3. Experiment 3 The model revealed a significant effect of observation period (χ21 = 119.338, P < 0.0001), but not significant effect of conditioning cues (χ21 = 0.001, P = 0.992). Larvae conditioned with alarm cue plus catfish odour showed on average similar activity compared to larvae exposed to control water plus catfish odour during the conditioning 5
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Fig. 3. Frequency of entering the centre of the arena of (a) 12-dpf and (b) 24-dpf zebrafish in each minute of experiment 1 and proportion of time spent in the centre of the arena (arcsine square root transformation) of (c) 12-dpf and (d) 24-dpf zebrafish in each minute of experiment 1. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue (either alarm cue, water control, or fish odour control; see colour legend).
pairing with alarm cue.
phase (Fig. 4b). The observation period × conditioning cues interaction was significant (χ21 = 51.485, P < 0.0001). Larvae conditioned with alarm cue showed a greater reduction in activity in the post-exposure observation period compared to larvae exposed to water during conditioning, as expected if larvae learned to recognise the novel odour via
4. Discussion Behavioural responses induced by alarm cues may notably favour 6
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Fig. 4. Activity (distance moved) of zebrafish in each minute of the testing phase of (a) experiment 2 and (b) experiment 3. Data points indicate means and error bars indicate SEMs. Dashed lines indicate the exposure to the cue in the testing phase (catfish odour).
characterising the ontogeny of zebrafish alarm cue at the chemical level. However, this is a difficult task because the composition of alarm cue is mostly unknown and probably consists of a mixture of many substances, with only some of them having been characterised yet (blood: [17]; chondroitin: [16]; hypoxanthine 3-N-oxide [14]:). For researchers interested in using alarm cue response as a model, our results suggest to test zebrafish aged approximately 24 dpf and to avoid the use of younger subjects, which can show a weaker response. The results of experiment 1 indicate that zebrafish larvae are sensitive to the alarm cue of conspecifics of the same age, which can pave the way to developing models in several fields of translational research. Future studies should try to understand the mechanisms causing the alarm cue-induced behavioural responses and the effects of the exposure to alarm cue in zebrafish larvae. The first point to be addressed is the identity of the molecules perceived as alarm cue by larvae. Indeed, we obtained alarm cue by homogenising the entire donor, as commonly due to study small species and early developmental stages [29,54]. Literature suggests hypoxanthine-3 N-oxide [12–14] and glycosaminoglycan chondroitin [16] as candidate molecules, although most authors recognise alarm cue to be a mixture of chemicals (reviewed in Chivers et al. [20]). The second relevant point to address is which neural partway controls alarm cue response. In adult zebrafish, the response to alarm cue has been mainly characterised using pharmacological methods. The serotonergic system seems to be involved because the brain of adult zebrafish exposed to alarm cue shows increased extracellular 5-HT levels [40]. Cholinergic signalling is also involved because nicotine blocks the effects of alarm cue in adult zebrafish [55]. In adult zebrafish, alarm cue also causes an increase in blood haemoglobin, glucose, epinephrine and norepinephrine levels [40]. Some studies tried to identify the neural substrates responsible for reaction to alarm cue. In the crucian carp, Carassius carassius, surgical ablation experiments have revealed that alarm cue reaction is mediated by sensory neurons making synaptic connections with the medial bundle of the medial olfactory tract [56]. Mathuru and colleagues [16] reported that in adult and 28–50 dpf zebrafish, skin extracts activated
research on zebrafish anxiety, fear, stress and cognitive functions such as learning. This contribution might also be relevant for research on larvae, but their behavioural response to alarm cues has not yet been documented. Here, we provided evidence that zebrafish larvae exhibit innate response to alarm cues obtained by homogenising conspecific larvae. In addition, zebrafish larvae could be conditioned by pairing these alarm cues with a novel odour. In experiment 1, we exposed 12- and 24-dpf zebrafish to conspecific alarm cue to study their innate behavioural response. We found reduced activity among the larvae exposed to the alarm cue, stronger than that due to the experimental manipulation and the exposure to a novel fish cue, as suggested by the comparison with the control groups. Similar conclusions were reached considering one measure of thigmotaxis—the frequency of entering the centre of the arena—though the effects were substantially less clear than those obtained from activity. The second measure of thigmotaxis, the proportion of time spent in the centre of the arena, was correlated with the activity but did not evidenced the alarm cue’s effect. Therefore, it seems reasonable that the activity is more appropriate for studying alarm cue responses in larvae. The analyses on activity separated per age suggested that the larvae responded to alarm cue at both 12 and 24 dpf. However, the response to the alarm cue was much stronger in 24-dpf compared to 12-dpf zebrafish, as indicated by the significant three-way interaction and by plotting (Fig. 2). There are at least two possible explanations for the age difference. First, larvae in the younger group might not have fully developed the nervous substrates necessary to detect and recognise alarm cue, which might cause a weaker response. The number of neurons in the zebrafish olfactory bulb after hatching is lower compared to that of adults (∼5 %) and subsequently increases with ontogeny [50,51]. On the other hand, the alarm cue of young larvae may be produced in smaller quantities, and the response to alarm cue is often dose dependent [52] and/or the alarm cue of young larvae is less efficient in evoking the response. For example, male fathead minnows, Pimephales promelas, do not produce active alarm substances during the breeding season [53]. The age difference might be further investigated by
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based on other forms of associative learning usually require extensive and time-consuming trainings [60]. On the other hand, some characteristics of predator recognition learning might represent confounding factors and should be carefully considered. In contrast with other forms of learning, multiple conditioning events with alarm cue might be detrimental. When risk persists for a considerable amount of time, prey often dramatically reduce responses to the predator, to counterbalance the loss of resources due to antipredator behaviour, such as hiding, which occurs during the initial exposure to the risk (risk allocation: [61]). Similarly, it is difficult to assess memory window for predator recognition learning, although this theoretically requires little change in the procedure that we adopted to study learning. Indeed, prey might stop responding to a predator cue even if they still recognise it as dangerous. Another possible limitation regards discrimination learning because prey tend to respond to olfactory cues somehow similar to that used during conditioning (generalization of predator recognition [46]). In our study, we used a wild-type outbreed strain of zebrafish, whereas most of the translational research exploits inbreed and genetically modified lines. It should be verified whether in other zebrafish strains, larvae’s response to alarm cues is similar to that observed in this study. However, we do not expect large differences between strains. The main variable measured in this study, activity reduction, is highly reliable and showed by almost all fish species investigated (e.g., Ferrari et al. [10]). Among adults, different strains respond differently to alarm cues [41,62], but these differences were usually limited to behavioural measures other than activity reduction (e.g., number of vertical drifts, erratic movements, and in angular velocity). In conclusion, our study revealed that zebrafish larvae exhibit direct behavioural responses to alarm cue and use it to develop a learned antipredator response. Recent studies with a similar experimental approach but performed with adult zebrafish are unravelling how alarm cue responses vary by the context, the paradigm and the population of fish used and how alarm cue modulates aggression, the monoaminergic system and spatial behaviour [62–64]. It will be interesting to investigate these factors in larvae to further evaluate the possibility of developing behavioural and cognitive models based on zebrafish at the larval stage. These models might benefit from being quick to perform and exploiting the zebrafish’s natural antipredator behaviour.
the mediodorsal posterior olfactory bulb, which has a projection to the habenula. A similar activation was reported for 3-week-old zebrafish, despite the lack of a behavioural response to the substance. These data suggest that the response of larvae can be mediated by the same neural substrates as for adults. The c-fos expression analyses and the wholebrain imaging techniques available for freely swimming larvae [7,57] might provide important advancements for understanding the neurons involved in alarm cue response. Experiment 2 was aimed at studying whether 24-dpf zebrafish could be conditioned to recognise a novel odour as dangerous via pairing with alarm cue. In case of learning, larvae were expected to show an antipredator response (activity reduction) to the conditioned odour administered in testing phase. When considering the entire testing time, we found no evidence of a learned antipredator response in larvae conditioned with alarm cue plus a novel fish odour: their response was not detectably different from that of control larvae exposed to water during the conditioning phase. Larvae might not be able learn to recognise a novel odour as dangerous via pairing with alarm cue, even if experiment 1 provided evidence that they can perceive the alarm cue. A similar ontogenetic effect on cognitive abilities has been reported for another small teleost fish, the guppy, Poecilia reticulata. Individuals of this species do not reach their maximum accuracy in a cognitive task until the age of 40 days [58]. However, an analysis restricted to the minute before and after cue administration revealed a slightly stronger response to the novel fish odour, in terms of activity reduction, in larvae conditioned with alarm cue, as compared to controls exposed to water during the conditioning phase. This effect leads to a second, methodological explanation. We might have failed to detect the learned antipredator response because this effect was masked by other factors. Larvae conditioned with alarm cue had much lower activity in the test phase, even before the exposure to testing cues (Fig. 4a), as compared to larvae exposed to water during conditioning. This lower activity was probably a mechanism of dealing with the risk of predation and went on for up to 6 h after the conditioning phase (i.e., when the test phase took place). To verify this interpretation, we ran experiment 3 using a greater interval between the conditioning and the test phase (24 h). In experiment 3, we did not observe reduced baseline activity among larvae exposed to alarm cue (Fig. 4b). Also, the effect of the learned antipredator response was detected. Therefore, experiments 2 and 3 provided evidence that zebrafish larvae at the age of 24 dpf can learn to recognise a novel predator cue when it is paired with alarm cue, but that this effect is clearly visible only with an extended interval between conditioning and testing. To date, paradigms for studying cognition in zebrafish larvae are mostly based on simple forms of learning, such as habituation and sensitisation [59]. Alarm cue-based learning might allow developing paradigms to study more complex cognitive abilities, such as associative learning. Beside training larvae to recognise a novel odour, it might be possible to train them to associate danger with a specific visual cue and to study visual discrimination learning. For example, larvae can be exposed to two objects, including one associated with alarm cue, allowing researchers to test whether larvae can distinguish between the two objects. It may also be possible to use alarm cue to study spatial learning. Brown (2003) [8,9] presented rainbowfish, Melanotaenia spp., with a predator model placed in a particular microhabitat, and the fish learned to avoid the ‘dangerous’ microhabitat. A similar learning process might be triggered by alarm cue instead of a predator model and be used to investigate spatial abilities in larval zebrafish. It may be also possible to assess mnemonic retention because the association between a cue and the danger usually lasts for many days [48]. Using these and similar paradigms might allow for assessing learning differences between larvae with different genotypes or exposed to different pharmacological treatments, with two great advantages. First, complex cognitive processes can be studied by measuring a rather simple behavioural response (activity reduction). Second, experiments with these paradigms would require only one training session, whereas paradigms
CRediT authorship contribution statement Tyrone Lucon-Xiccato: Conceptualization, Methodology, Formal analysis, Funding acquisition, Writing - original draft. Giuseppe Di Mauro: Conceptualization, Data curation, Writing - review & editing. Angelo Bisazza: Conceptualization, Funding acquisition, Writing - review & editing. Cristiano Bertolucci: Conceptualization, Funding acquisition, Writing - review & editing. Acknowledgements We have no competing interests. This work was supported by FAR2018, FAR2019 and FIR2018 from University of Ferrara to TLX and CB, by DOR grant from Università di Padova to AB, and by 31 st PhD program in Evolutionary Biology and Ecology at the University of Ferrara. References [1] R.J. Egan, C.L. Bergner, P.C. Hart, J.M. Cachat, P.R. Canavello, M.F. Elegante, et al., Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish, Behav. Brain Res. 205 (2009) 38–44. [2] S. Jesuthasan, Fear, anxiety, and control in the zebrafish, Dev. Neurobiol. 72 (2012) 395–403. [3] B.V. Hope, T.J. Hamilton, P.L. Hurd, Submerged plus maze: a novel test for studying anxiety-like behaviour in fish, Behav. Brain Res. 362 (2019) 332–337. [4] C. Maximino, T.M. de Brito, A.W. da Silva Batista, A.M. Herculano, S. Morato,
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