Social learning is an important mechanism for acquiring knowledge about environmental risk. However, little work has explored the learning of safety and how such learning outcomes are shaped by the social environment. Here, we exposed minnows, Pimephales promelas , to a high-risk environment to induce behavioural responses associated with fear (e.g. neophobia). We then used the presence of calm conspecific models (low-risk individuals) to weaken these responses. When observers (individuals from the high-risk environment) and models were paired consistently in a one-on-one setting, the observers showed no recovery (i.e. no weakening of the fear responses), and instead the models indirectly acquired those responses (i.e. a socially transmitted state of fear). However, observers paired with models that were periodically replaced with new calm models showed a significant recovery, and each new model showed diminished socially transmitted fear. We argue that our understanding of predation-related fear and social information transfer can prove fruitful in understanding problems with fear and stress across animal taxa, including among humans who experience post-traumatic stress and secondary trauma. Our findings indicate that the periodic replacement of models can promote fear recovery in observers and reduce socially transmitted fear in models.

1. Introduction

Living in groups can provide many fitness benefits such as increased foraging efficiency, increased repulsion of enemies and increased transfer of information [1]. Such social information transfer can be an efficient way to gain knowledge without direct exposure to danger [2]. Within a social group, a higher number of knowledgeable individuals is thought to increase the chances for naive observers to acquire that knowledge. This pattern is found in the context of task performance, where an increased number of experienced individuals (hereafter, models) correlates with more accurate and efficient responses (e.g. [3]). Likewise, the social transfer of foraging information is also enhanced by the presence of more models [4]. For example, Reebs [5] found that the ‘following’ behaviour of observers undertaking a foraging task was stronger when the model-to-observer ratio was higher.

The risk of predation has a pervasive influence on prey, often causing long-term changes in neurophysiology, morphology and behaviour [6]. Learning about predation risk via social information is widespread across animal taxa (reviewed in [2]), with some research exploring how such learning is influenced by group size [7–10]. For example, tadpoles, Lithobates sylvaticus, conditioned with a higher model-to-observer (5 : 2) ratio learned to respond to a specific predator with a greater fear intensity than those conditioned with a lower ratio (2 : 5) [9]. Hence, more models increased the social transfer of information about risk. However, much less is known about how groups play a role in the transfer of information about safety from predators. Error management theory predicts that individuals which are uncertain should over-respond to predation threats because the cost of being wrong is so high [11]. Hence, we would expect the social transfer of information about safety to require more reinforcement than the transfer of information about risk. Indeed, overriding previously learned safety with social information about risk can occur from a one-time experience with a single model [12]. However, previously learned risk can be much more difficult to override socially. In a one-on-one observer-to-model setting, the observer and model can influence each other in a contra-directional fashion, with the observer having an opportunity to learn safety from an experienced model, and the model concurrently learning risk from the observer [13,14].

In a high-risk environment, fathead minnows, Pimephales promelas, like many species, acquire long-lasting behavioural responses associated with fear such as neophobia (reviewed in [15]). Moreover, this generalized state of fear can be indirectly acquired by social companions in the absence of direct exposure to danger (i.e. socially transmitted neophobia) [13,14]. In one study with fathead minnows (hereafter, minnows), high-risk observers (i.e. those from a high-risk environment and not in the sense of risk taking) interacted with different sized groups of calm conspecific models (i.e. individuals from a low-risk environment that were swimming calmly). A large group of social models influenced observers to behave as though the environment was now safe [16], via a mechanism known as ‘social buffering’ [17]. Hence, like the social transfer of information about risk, more models also facilitated the social transfer of information about safety.

Within a social group, observer individuals sample information from other group members, often based on nearest-neighbour distances [18,19]. Previous studies on the effect of group size on the social transfer of predation information have assessed situations where all group members were together at one point in time (e.g. [7,10]) rather than the way each piece of social information influenced an initially naive observer. Whether separate encounters with individual models might weaken fear in observers over time, similar to encounters within a group, remains unclear. In such a scenario, the absence of information transfer among calm models could make them more susceptible to socially transferred fear from observers.

Our goal in this study was to assess the impact of the removal and subsequent replacement of the model on the fear recovery of high-risk minnows (i.e. the weakening of the behavioural responses associated with a high-risk environment). In addition, we wanted to closely monitor how models were affected by observers, and thus we tested both observers and their models, including those that were replaced. We hypothesized that, as in previous work, models would experience a socially transmitted state of fear, and thus become poorer models for demonstrating safety and facilitating the recovery of observers. However, we expected that after model replacement, the new replacement models would better demonstrate safety to high-risk observers compared to previously affected models. Therefore, the periodic replacement of models should benefit the recovery of observers, and likewise, new replacement models should be less fearful than the models they replaced. Hence, we predicted that: (i) observers paired with replacement models would show decreased fear responses (e.g. more time freezing and less time swimming calmly) compared to those paired consistently with models, and (ii) that the final replacement model would show lower levels of fear than models paired consistently with observers. Alternatively, the change from a familiar model to a new model could be so stressful that it could prevent any recovery of observers. In this case, we would predict that observers paired with replacement models would show the strongest fear responses. In our discussion, we present a case where the field of animal behaviour, and specifically social learning theory, can inform and provide insights into factors affecting the fear severity and the potential for overcoming such fear, with specific attention on post-traumatic stress (PTS).

2. Material and methods

(a) Fish and cues

The fathead minnow is a fish species that spends the majority of its time engaged in social interactions, living in large social groups referred to as ‘shoals’ [20]. They do not typically make synchronized movements but have occasionally been observed to do so when avoiding danger ([21]; A.L. Crane 2015, personal observations). During the reproductive season, males display social competition for mates while defending territories, and after reproduction the males defend the fertilized eggs from predators, including conspecifics [22,23]. This species has been particularly well studied in the context of social learning of predation risk (e.g. [12,24]).

We collected minnows from Feedlot Pond in central Saskatchewan where they are exposed primarily to avian and invertebrate predators but have no fish predators. After capture, we transferred minnows to two laboratory pools (2460 l) at a density of approximately 300 individuals per pool. Each pool received filtered flow-through water and aeration and contained synthetic plant-like structures. All individuals were given four months of acclimation to the pools and were behaving calmly before the study began.

Like many other fishes, minnows have an alarm substance contained in their skin, described originally as ‘Schreckstoff’ by von Frisch [25,26]. This substance is released when the skin is physically damaged by a predator, thus being a reliable ‘alarm cue’ indicating predation risk. Nearby conspecifics (and in some cases heterospecifics) can detect alarm cues via olfaction whereby they innately recognize the risk in their environment. We used standard procedures for making alarm cues, euthanizing five individuals with a blow to the head in accordance with the Canadian Council on Animal Care [27]. Skin was homogenized and diluted to reach an ecologically relevant concentration (1 cm2 of skin in 40 l of water) that is known to elicit a fear response [12,28]. Repeated exposure to alarm cues at this concentration induces behavioural responses (e.g. neophobia) that are associated with a high-risk environment [13,14].

We obtained novel odour from two Lake Sturgeon, Carpenter fulvescens, a species that is allopatric to the minnows in our study, by housing two individuals in unfiltered water for 24 h at a standardized concentration (100 ml g−1 of fish). In the wild, these sturgeon feed on benthic invertebrates, but the individuals used in this study from our laboratory stock colony were raised on a diet of sinking pellets. Previous work has shown that exposure to novel sturgeon odour does not cause a fear response in minnows from this population when maintained under low-risk conditions [13].

(b) Phase 1: background regime for high-risk individuals

Throughout the experiment, fish were housed in 37 l aquaria with filtered water, aeration, gravel and shelter objects. In the first phase of the experiment, minnows that would serve as high-risk observers were repeatedly exposed to alarm cues (3× per day with 2–3 h between exposures) in 23 different groups of four individuals (figure 1). We chose to expose fish in groups based on our previous work showing that fear in groups (i.e. socially reinforced fear) was difficult to override via one-on-one model therapy [16]. The exposures occurred by gently injecting alarm cues (5 ml) with a syringe that was connected to a 1.5 m hose attached to an air stone in the tank. After 4 days of exposure, the risk period ended, and minnows were transferred to new tanks for the conditioning phase. Figure 1. Experimental phases for risk-exposed observers (labelled ‘O’) and their models (labelled ‘M’). First, observers experienced risk from 12 exposures to alarm cues in a group of four fish. Then, observers had the opportunity to interact with calm models, except in the ‘unpaired’ control group where observers and models were not paired. The replacement group differed from the consistent group by experiencing the removal and replacement of the model every 3 days. Testing with novel odour occurred 1 day following the conditioning period for observers, whereas all models were tested 1 day after their removal from the observer's tank.

(c) Phase 2: post-risk pairings with models

During a 9-day period, two treatment groups consisted of high-risk observers that were individually paired with a single calm model. These models were individuals that were moved directly from the holding pools (i.e. no alarm cue exposures) and were qualitatively assessed as behaving calmly. After the pairings, each model either remained in the tank for the full 9 days (a consistent pairing), or the model was removed and replaced by a new low-risk model every 3 days (replacement pairings). Models were always removed gently with a dip net. To control for this disturbance across the treatment groups, we handled the other models identically to the replacement models every 3 days, but instead of removing models as in the replacement pairings, they were immediately put back into the same tank. At the end of the 9 days, all models were moved individually into their own tanks, whereas observers remained in the same tank for testing.

As a control group, other high-risk observers were kept separately from calm models (the ‘unpaired’ group) (figure 1), allowing us to assess whether observers and models simply changed their responses over time, or whether they changed behaviour owing to the model-observer pairings. However, keeping these ‘unpaired’ observers isolated during a 9-day period would have been an additional stressor on this treatment group, so we chose to add another high-risk individual (a non-focal fish) to interact with the ‘unpaired’ observers. Hence, we controlled for the presence of another fish, and thus, ‘unpaired’ observers were only unpaired in regard to the calm models (figure 1). Likewise, in a separate tank, each model in the ‘unpaired’ treatment was paired with another calm individual, again to avoid a confounding stressor from isolation. For this treatment, each non-focal observer and a focal model were disturbed in the same manner as the models in the consistent treatment.

(d) Phase 3: testing for recovery

One day following the social conditioning period, observers and models were tested separately (figure 1), allowing us to assess their behaviour alone without the influence of other individuals. The back and sides of each tank were covered with an opaque plastic film to block visual cues among tanks. We also covered the front of tanks with a translucent film (5% visual light transmission) that, coupled with lighting above each tank, provided us with a clear view of the fish while minimizing our visual cues. Testing was conducted blind by a single human observer. During this testing period, minnows were first observed during an 8 min pre-stimulus period where we recorded their time spent pacing (crossing greater than 6 cm s−1 in a route-tracing pattern) and their time spent freezing (centre of the body not moving). Then, we injected the novel odour (20 ml) and recorded data for an additional 8 min to assess any neophobic response from behavioural changes between the pre- and post-stimulus periods. For each trial period, the total duration (480 s) minus the time spent pacing and freezing was expressed as the time spent swimming calmly. Calm swimming consisted of swimming continuously, often 1–5 cm above the substrate while searching for food, which is a typical display by this species in the absence of risk [29,30]. Minnows from high-risk backgrounds, however, display a higher proportion of time spent freezing and pacing [14], which is a stereotypy often seen among captive individuals that are highly stressed [31,32]. We also recorded data on the replacement models that were used at each time period (days 1–3, days 4–6 and days 7–9), whereas observers were only tested after the full 9-day period.

(e) Statistical analysis

We analysed observers and the final models separately after the 9-day period, as they were not directly comparable owing to the procedural difference of models being moved into a new tank with only 1 day of acclimation, whereas observers remained in their tank to which they had acclimated for 10 days. We used MANOVAs with the proportion of freezing time and time spent swimming calmly as response variables (the proportion of pacing time was omitted because of collinearity) and the treatment (unpaired, consistent or replacement) as a fixed factor. For data from observers, we included the background tank as a random factor (nested model with Type I sum of squares) to account for risk exposures occurring in groups and hence fish in each group not being independent. First, we analysed the pre-stimulus data alone to gauge baseline activity, with post-hoc MANOVAs comparing specific groups (consistent versus unpaired and replacement versus unpaired). We then assessed neophobic responses by using repeated-measured MANOVAs with treatment as a fixed factor and time (pre- versus post-stimulus period) as the within-subjects factor, and again the background tank as a random factor for observers. We assessed differences in neophobia by using time × factor interactions, depicted graphically by differences in slopes. Lower levels of baseline freezing and neophobia were considered as ‘calmer’ behaviour. Finally, we used this same overall approach to assess the behaviour of replacement models from each conditioning period (days 1–3, days 4–6, days 7–9). Due to a large number of terms in our MANOVA models, we provide only p-values of interest in the results and report all F, df, and p-values in electronic supplementary material, Tables S1–3. We report p-values for Pillai's Trace owing to its conservativeness [33], and with α = 0.05. Analyses were conducted in SPSS 20.

3. Results

(a) Recovery for observers via replacement models

Baseline activity significantly differed among the treatments (p = 0.004, figure 2a,c). Compared to control observers (i.e. the high-risk unpaired observers), observers were calmer following replacement pairings (p = 0.006), but not following a consistent pairing with the same model (p = 0.22). Neophobic responses also differed across treatments (p = 0.002, figure 2a,c). Consistently paired observers became more neophobic (p = 0.008), while observers paired with replacement models showed overall calmer behaviour during both the pre- and post-stimulus periods (overall main effect: p = 0.001; interaction: p = 0.59). Figure 2. Mean (±s.e.) proportional time spent freezing (a,b) and time spent swimming calmly (c,d) for observers (a,c) and models (b,d) tested alone, before (pre) and after (post) exposure to a novel odour. Observers had prior experience with a high-risk environment. Then, during a 9 day conditioning phase, high-risk observers were paired with calm models: either consistently, with the model replaced every 3 days (replacement), or the observer and model were not paired (unpaired). Testing of observers occurred 1 day following the conditioning period, and for models 1 day after their removal from the tank. Steeper slopes represent stronger behavioural changes in response to the odour. Sample sizes ranged from 18 to 32 per group. Data for pacing are presented in the electronic supplementary material, figure S1.

(b) Socially transmitted fear in models

As expected, models were influenced by their observers (baseline: p = 0.029; neophobia: p = 0.017, figure 2b,d). At the end of the pairings, the final replacement models behaved similarly to the low-risk control (unpaired) models (baseline: p = 0.66; neophobia: p = 0.77), whereas the consistently paired models acquired high-risk responses from their observers (baseline: p = 0.017; neophobia: p = 0.006). However, replacement models behaved differently depending on the timing of their pairing with observers (baseline: p < 0.001; neophobia: p = 0.001, figure 3a,b), with a pattern of becoming calmer over time. Compared to the first model, the second model was significantly calmer (baseline: p = 0.005; neophobia: p = 0.021), and the final model appeared to continue this trend, although not significantly (baseline: p = 0.70; neophobia: p = 0.18). Figure 3. Mean (±s.e.) proportional time spent freezing (a) and time spent swimming calmly (b) for models tested individually both before (pre) and after (post) exposure to a novel odour. Steeper slopes represent stronger behavioural changes in response to the odour. Replacement models were paired with high-risk observers on days 1–3, 4–6 or 7–9 following the observers' risk exposure. Sample sizes were 32 per group. Data for pacing are presented in the electronic supplementary material, figure S2.

4. Discussion

The replacement of models that had experienced socially transmitted fear facilitated a significant and modest recovery for high-risk observers, whereas the high-risk behaviours of observers appeared unaffected when their models were not replaced (i.e. the consistently paired treatment). The difference in recovery between the replacement pairings and the consistent pairings appeared to result from each new low-risk model slightly diminishing the observer's high-risk responses. Although we did not test observers following each replacement period, the first model experienced a high level of socially transmitted fear, with subsequent models experiencing significantly less (figure 3). While our previous research demonstrated that a group of calm models was effective in reducing fear in high-risk observers [16], interacting with different individual models in succession (this experiment) was a fundamentally different situation because the models never interacted with one another. Hence, our work has revealed that both a group of calm models together and multiple models in individual encounters can cause high-risk observers to show less fear in their environment.

Socially transmitted fear was significant for models that were consistently paired with high-risk observers, but overall, the level of socially transmitted fear appeared weaker than the directly acquired phenotypes in observers, especially in terms of freezing (figure 2a,c versus figure 2b,d). Although models and observers were not directly comparable because models were moved into new tanks while observers were not, this confounding factor (a stressful disturbance) should have affected models more than observers. Thus, in this scenario, socially transmitted fear appears less intense than the effects of direct danger, as documented in previous studies [13,14].

Our results probably have implications for managing fear among social animals housed in animal facilities such as zoos, hatcheries, public aquaria and research laboratories. Common methods for reducing stress in captive animals include providing environmental enrichment, medication, exercise and training (e.g. [34,35]), but to our knowledge social buffering has yet to receive much consideration. For instance, newly collected and/or transported individuals may benefit (i.e. experience a faster reduction in stress) from observing calm individuals. Even if animals cannot fully interact owing to quarantine, they may still benefit from visual or auditory cues from calm individuals. The numbers of stressed and calm individuals should be carefully balanced to accelerate returns to calmness and prevent ‘ripple effects’ in terms of socially transmitted fear. Our results indicate that higher ratios of calm-to-stressed individuals should be more effective, whereas isolating stressed individuals will slow or prevent recovery.

Behavioural problems related to fear are also commonly seen in companion animals like domestic dogs and cats [36]. In dogs, aggressiveness, separation fear and a generalized state of fear are common [37]. Generalized fear is often seen in abused individuals [38], those living in shelters [39] and those with working roles such as guarding and medical alert, where fear recovery is critically important [40]. Standard medications are often used to reduce a fearful state [36], as is artificial selection for individuals with fewer fear problems [40]. We are not aware of any studies that intentionally use intraspecific social therapy for companion animals. However, some studies on dogs have used a conspecific chemical cue (‘dog-appeasing pheromone’ secreted during lactation) to reduce fearful behaviours such as vigilance and pacing [39,41]. Rooney et al. [40] argued that future research on fear recovery should explore new methods involving social learning, stressing that the influence of calm conspecific models needs to be evaluated.

Across taxa, exposure to dangerous events can have remarkably similar effects on neurobiology and behaviour [6,42,43]. While we conducted this study to better understand social learning of risk and safety in minnows, we also sought to apply experimental outcomes to the recovery of fear-related problems in humans. Animal models have also made a large contribution to our understanding of human fear problems such as PTS, characterized by fearful responses such as withdrawal, hypervigilance and fear of the unexpected [44,45]. We argue that the field of animal behaviour, and specifically predation ecology and social learning theory, can inform and provide insights into factors affecting such fear and the potential for recovery.

Even fishes have shown strong potential for exploring dimensions of PTS, with high face validity—exposure to elevated levels of risk can induce symptoms that are similar to the responses associated with PTS (reviewed in [16,46]). These responses can also be socially transmitted, like in humans, where the term ‘secondary trauma’ is used to encompass this general phenomenon [47,48]. In humans, more time spent interacting with direct trauma victims appears to cause more severe secondary trauma in therapists (e.g. [49,50]). We still know little, however, about how secondary trauma compares with the symptoms of the direct trauma victims, or when and for how long the acquired symptoms persist [51,52]. Steps to prevent and overcome secondary trauma have received some attention, for instance, ceasing interaction with the trauma victim and adopting self-care strategies. However, a recent literature review on alleviating secondary trauma found that no randomized experimental (or quasi-experimental) studies have been conducted, and the authors viewed the observational studies as being plagued by small sample sizes [53].

Here, our experiment modelled how the periodic replacement of therapists that are experiencing secondary trauma might reduce the trauma symptoms of both themselves and their patients. Indeed, the social environment appears critical for the susceptibility and recovery of PTS [54]. In our view, our understanding of the social ecology of PTS and behavioural therapy can be furthered by research on non-human animals. Even fishes may provide insights. Their abundance can facilitate complex and controlled experiments with robust sample sizes. Such studies can either fortify or challenge our current views on PTS and potentially stimulate new ideas for recovery strategies.

Ethics

All procedures were conducted in accordance with the Canadian Council on Animal Care guidelines and were approved by the University Council on Animal Care (protocol 20130079) of the University of Saskatchewan. Minnows were collected under a Saskatchewan Ministry of the Environment Special Collection Permit (SPC17-AR03).

Data accessibility

Data from this research are freely available in the Dryad Digital Repository: https://www.datadryad.org/resource/doi:10.5061/dryad.r3117t8 [55].

Authors' contributions

A.L.C. and M.C.O.F. developed this research. A.L.C. conducted behavioural trials and analysed the data. K.R.B.-N. and L.H.S. conducted background risk exposures. All authors contributed to writing the manuscript.

Competing interests

We have no competing interests.

Funding

Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada and the Animal Behaviour Society.

Footnotes

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.4238558.