Research reportAlarm cue-mediated response and learning in zebrafish larvae
Introduction
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], [9], [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], [9], [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 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], [22], [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], [9], [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 large-scale screenings of drugs and genotypes. The innate behavioural 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], [30], [31], [32], [33], [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 24-dpf 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 cue-mediated 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.
Section snippets
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,
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 (
Discussion
Behavioural responses induced by alarm cues may notably favour 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
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.
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