Simulating more realistic predation threat using attack playbacks

Introduction

Predation risk is thought to play an important role in determining life-history trajectories, the social system and behaviours among animals (Holling, 1965; Tollrian, 1995; Stearns, 2000; Heg et al., 2004; Preisser & Bolnick, 2008; Walsh & Reznick, 2008; Groenewoud et al., 2016; Fischer et al., 2017a). While an obvious way of testing the impacts of predation risk is to manipulate the number of predators and/or to present predators to potential prey (e.g., Chiverton, 1986; Fontaine & Martin, 2006), such experiments are not always logistically or ethically feasible. An alternative approach is to use ‘virtual proxies’ on captive prey, which reduces the ethical implications of subjecting such prey to live predators and removes the logistical and ethical issues of manipulating predators. Many proxies such as decoys (Arroyo, Mougeot & Bretagnolle, 2001; Li et al., 2014; Catano et al., 2016; Griesser & Suzuki, 2017; Morales, Lucas & Velando, 2018), screen presentations (Johnson & Basolo, 2003; Fischer et al., 2014; Scherer, Godin & Schuett, 2017; Watve & Taborsky, 2019) or kairomones and alarm cues (reviewed in Ferrari, Wisenden & Chivers, 2010) have been used successfully so far. Nevertheless, the utility of virtual proxies rests with their ability to replicate the natural predator–prey encounter. Prey may use multiple cues to detect a predator (Gonzálvez & Rodríguez-Gironés, 2013) or multiple cues may have a different effect than a single cue (Hartman & Abrahams, 2000; Hickman, Stone & Mathis, 2004; Martin et al., 2010; Ward & Mehner, 2010). Further, prey may use specific features of the predator’s behaviour or orientation to assess the immediate risk and fine-tune their behaviour (Kent et al., 2019). Most importantly, live predators and prey will naturally interact, an element inherently lacking in the use of proxies. Despite this, such interactions are rarely incorporated into experiments using virtual proxies (but see Hartman & Abrahams, 2000)—increasing the chances that prey will quickly habituate or even ignore the stimulus (Holomuzki & Hatchett, 1994; Raderschall, Magrath & Hemmi, 2011). To minimize these shortcomings, it is necessary to ensure that virtual proxies (i) stimulate the relevant sensory modalities and (ii) provide salient features of natural predators eliciting ecologically relevant prey responses.

In aquatic systems, the role of visual as well as chemical cues in predator detection and response is well studied. Chemical cues released from damaged tissues of injured conspecifics (‘alarm cues’) play an important role in detection of predators in many species and are used in experimental manipulation (e.g., Brown & Smith, 1998; Hartman & Abrahams, 2000; Brown, 2003; McCormick & Manassa, 2008; Ferrari, Wisenden & Chivers, 2010; Manassa & McCormick, 2012; Moscicki & Hurd, 2017; Lucon-Xiccato, 2019). For example, skin extract from conspecifics administered along with predator odour helps fishery-reared rainbow trouts to recognise predators (Brown & Smith, 1998). Similarly, the use of visual proxies in aquatic systems is also well-established (e.g., Johnson & Basolo, 2003; Gerlai, 2013; Fischer et al., 2014; O’Connor et al., 2015; Dieng et al., 2017). While most studies report a successful use of proxies in inducing predicted anti-predator response, none have compared their effectiveness to live predator presentations.

Neolamprologus pulcher is a cooperatively breeding cichlid from Lake Tanganyika that lives in groups of up to 25 individuals (Taborsky, 2016). The social structure of N. pulcher is driven primarily by predation pressure (Groenewoud et al., 2016) and thus, manipulation of predation threat has been often employed to study their social dynamics (e.g., Heg & Taborsky, 2010; Fischer et al., 2017a; Watve & Taborsky, 2019). Turbidity and water quality in the lake differs between populations (Plisnier, 2002) and between seasons (Plisnier et al., 1999). Therefore, it is expected that N. pulcher would rely on both visual and chemical cues flexibly. Lepidiolamprologus elongatus is a common and ferocious predator of N. pulcher that is most often used in manipulation experiments in field as well as laboratory. It has been shown that N. pulcher can successfully distinguish images of L. elongatus from herbivorous cichlids of the same size and respond accordingly (Fischer et al., 2014). Chemical cues from L. elongatus alone can also induce appropriate anti-predator responses in very young fry of N. pulcher (Fischer et al., 2017a). Predator recognition in N. pulcher seems to be innate, as both these studies observed anti-predator response by naïve individuals. One study showed an unexpected reduction in response towards visual predator cues in the presence of conspecific skin extract on the anti-predator response of N. pulcher (O’Connor et al., 2015). It is, however, unknown if and how a combination of both predator odour and conspecific skin extract influences anti-predator response of N. pulcher.

Here we manipulated visual proxies to include prey-directed aggression by the predator L. elongatus to simulate a live interaction. We further tested the effect of predator odour and conspecific skin extract independently and in combination with each other on anti-predator responses of N. pulcher. We expected playbacks simulating directed attacks to induce responses more similar to a live predator but for lateral playbacks of normally swimming predators to induce lower responses than a live predator. We also predicted that N. pulcher will show a heightened response towards the predator visuals when predator odour was administered in conjunction with conspecific skin extract.

Methods

Experimental animals and housing conditions

The experiments were conducted at the Ethologische Station Hasli, University of Bern, Switzerland, under license BE-74/15 of Veterinary office of Kanton Bern. All experimental animals were obtained from the University of Bern laboratory stock, maintained in a 13:11 h light-dark cycle at 26 °C  ± 1 °C mimicking the conditions of Lake Tanganyika. All focal N. pulcher were predator-naïve adults (size range 40–45 mm) reared in the laboratory for 5 generations. All predators were first generation offspring of wild caught fish and were in the size range of 80–140 mm. The experimental 20-litre tanks (40 cm × 25 cm × 20 cm) were lined with 2 cm layer of sand and aerated by air stones. They were visually divided in three zones marked by pen on the front screen of each tank. The zone closest to the stimulus presentation was designated as ‘zone of interaction’, where the focal individual actively engaged in inspection of and defence against the stimulus. The farthest zone was considered as ‘safe zone’ and the time spent in this zone was taken as a measure of avoidance (Fig. 1). Each focal was tested in a separate experimental tank and was allowed to acclimatise overnight before testing. During acclimatisation, focal individuals were provided with ceramic pot halves as shelters, which were removed half an hour before each behavioural recording to avoid obstruction of observations. Visual playbacks were displayed on a 25 cm × 15 cm LED tablet screen (‘HP ElitePad 900’, resolution: 59 dot/cm) in both the experiments.

Results

Experiment 1

The number of aggressive displays towards the stimulus was determined by the type of presentation (GLMM, χ² = 23.41, p = 0.0001; Fig. 2A). Pairwise comparisons between presentation types revealed that there was no difference between the frontal playbacks and live stimulus (p = 0.13). However, lateral playbacks induced much lower aggression than live stimuli (p = 0.0008). Further, there was also a significant difference between the control stimulus and frontal playbacks (p = 0.02), which was not the case for lateral playbacks (p = 0.73 and the moving square (p = 0.98; Table 1, Fig. 2). Also the time spent being attentive to the stimulus was affected by the type of presentation (LMM, F = 4.14, p = 0.004; Fig. 2B). Focals were more attentive towards the live stimulus as compared to frontal playbacks (p = 0.02), the moving square (p = 0.004) and the control stimulus (p = 0.02), but not compared to lateral playbacks (p = 0.10; Table 2). No difference was found between lateral and frontal playbacks (p = 0.98; Table 2). Further, the time spent in the interaction zone was also influenced by presentation type (LMM power transformed, F = 5.34, p = 0.0009; Fig. 2C). Fish spent much longer in the zone of interaction when encountering a live predator compared to a blank control (p = 0.0007) or a moving square (p = 0.01) and marginally longer when compared to a lateral display (p = 0.09). Attention time did not differ between frontal playbacks and live predator (p = 0.46) or between lateral and frontal playbacks (p = 0.90; Table 3). Time in the safe zone was not affected by the type of display (LMM log-transformed, F = 1.29, p = 0.28; Fig. 2D).

Table 1:

Aggressive displays towards different types of predator presentations.

Pairwise comparisons between aggressive displays shown by focal N. pulcher (N = 16) towards different types visual predator presentations. All p-values presented are corrected for multiple comparisons using ‘mvt’ method. Significant p-values are marked with asterisks.

Display	Estimate	SE	df	t	p
Control-Square	−0.26	0.55	73	−0.47	0.98
Control-Lateral	−0.70	0.57	73	−1.22	0.73
Control-Frontal	−1.77	0.57	73	−3.08	0.02*
Control-Live	−2.98	0.62	73	−4.76	0.0001*
Square-Lateral	−0.43	0.56	73	−0.77	0.93
Square-Frontal	−1.50	0.56	73	−2.67	0.06.
Square-Live	−2.71	0.59	73	−4.56	0.0002*
Lateral-Frontal	−1.06	0.52	73	−2.05	0.25
Lateral-Live	−2.28	0.54	73	−4.17	0.0008*
Frontal-Live	−1.21	0.51	73	−2.35	0.13

DOI: 10.7717/peerj.8149/table-1

Table 2:

Attention towards different types of predator presentations.

Pairwise comparisons between time spent being attentive to different visual proxies by focal N. pulcher (N = 16). All p-values presented are corrected for multiple comparisons using ‘mvt’ method. Significant p-values are marked with asterisks.

Display	Estimate	SE	df	t	P
Control-Square	62.8	122	60	0.51	0.98
Control-Lateral	−80.9	122	60	−0.66	0.96
Control-Frontal	−13.9	122	60	−0.11	1.00
Control-Live	−384.4	122	60	−3.14	0.02*
Square-Lateral	−143.7	122	60	−1.17	0.76
Square-Frontal	−76.7	122	60	−0.62	0.96
Square-Live	−447.2	122	60	−3.66	0.004*
Lateral-Frontal	66.9	122	60	0.54	0.98
Lateral-Live	−303.5	122	60	−2.48	0.10
Frontal-Live	−370.5	122	60	−3.03	0.02*

DOI: 10.7717/peerj.8149/table-2

Table 3:

Time in interaction zone during different predator presentations.

Pairwise comparisons between time spent in interaction zone by focal N. pulcher (N = 16) during different visual proxies. All p-values presented are corrected for multiple comparisons using ‘mvt’ method. Significant p-values are marked with asterisks.

Display	Estimate	SE	df	t	P
Control-Square	−26.1	30.7	60	−0.85	0.91
Control-Lateral	−52.1	30.7	60	−1.70	0.44
Control-Frontal	−79.2	30.7	60	−2.58	0.08
Control-Live	−130.0	30.7	60	−4.24	0.0007*
Square-Lateral	−26.1	30.7	60	−0.85	0.91
Square-Frontal	−53.1	30.7	60	−1.73	0.42
Square-Live	−103.9	30.7	60	−3.38	0.01*
Lateral-Frontal	−27.1	30.7	60	−0.88	0.90
Lateral-Live	−77.9	30.7	60	−2.53	0.09
Frontal-Live	−50.8	30.7	60	−1.65	0.46

DOI: 10.7717/peerj.8149/table-3

Experiment 2

There was no effect of any chemical cue on the number of aggressive displays directed towards the screen (GLMM, χ² = 2.08, p = 0.55; Fig. 3A), on the time spent attentive to the screen (LMM, F = 1.15, p = 0.33; Fig. 3B), or on the time spent in the safe zone (LMM, F = 1.44, p = 0.24; Fig. 3C) or in the interaction zone (LMM, F = 0.36, p = 0.77; Fig. 3D).

Discussion

Here we tested the influence of different chemical and visual predator cues on the response of N. pulcher. The type of visual playbacks effected only those variables related to an interaction between the focal fish and screen, but it did not affect avoidance behaviour. Focals spent similar time in the interaction zone and showed similar aggression towards live stimuli and frontal playbacks, however, the response towards lateral playbacks was relatively weaker. Surprisingly, focals payed less attention towards the frontal playbacks than the live stimulus, whereas attention between the lateral playback and the live stimulus did not differ. The chemical cues did not affect the anti-predator behaviour of focals.

N. pulcher are sensitive to visual predator cues. For instance, they can distinguish heterospecifics, based simply on moving images (Fischer et al., 2014). However, images can fail to simulate a more realistic threat. Here we show that a playback of predator attack can induce a response similar to that shown towards a live predator. While prey species may recognize a predator based on images, they may not necessarily perceive them as threatening (Belin et al., 2018). Even for a moving visual, prey may only perceive the display as a threat if they include certain angles (Kent et al., 2019), postures (Etting & Isbell, 2014) or behaviours (Su & Lim, 2017) of the predator. Video playbacks have been used as predator cues in the past (Rowland, 1999; Carlile, Peters & Evans, 2006; Butler, Magrath & Peters, 2017). However, they lacked the element of prey-directed behaviour of the predator. We attempted to design visual predator cues that are as similar to an actual encounter as possible. While robotic models have been shown to hold potential (Krause, Winfield & Deneubourg, 2011; Ladu et al., 2015; Landgraf et al., 2016; Porfiri et al., 2019), they may not always be easily accessible. We propose a technically simple method of simulating a predator attack, which can be produced with ease and in a short time. Like other video playback, also frontal playbacks of attacking predators may eventually lead to habituations though. However, this can effectively be prevented by running them in an unpredictable temporal scheme (M Watve, 2019, unpublished data).

Even though the focal fish interacted with live stimuli and frontal playbacks, the apparent attacks from the predators in either case did not elicit a fear response in the fish. One possible explanation is that the predators never actually managed to harm the focals even during a live presentation, which may have emboldened the focals.

O’Connor et al. (2015) previously found conspecific skin extract to reduce the response towards a visual predator cue in N. pulcher, which is opposite to the prediction that skin extracts represent and alarm cue and should increase anti-predator responses (see Chivers & Smith, 1998 for comprehensive review). However, we did not find any such difference, neither when presenting skin extract alone or when paired with predator smell. Skin extract is assumed to contain alarm cues indicative of an injured conspecific, as shown in many other fish species including three-spine sticklebacks, Gasterosteus aculeatus (Brown & Godin, 1997) Rainbow trouts, Onchoryhnchus mykiss (Brown & Smith, 1997; Brown & Smith, 1998), red swordtails Xiphophorus helleri (Mirza, Scott & Chivers, 2001) and rainbow darters, Etheostoma caeruleum (Commens & Mathis, 1999), although this is not always the case. A recent study showed that muscle tissue extracts induce antipredator responses more effectively than skin or other tissue extracts (Meuthen et al., 2018) in another African cichlid P. taeniatus. It is highly likely that skin extract does not contain alarm substances in N. pulcher either. Further investigation is required to determine whether and to what extent alarm cues are present in other tissues of N. pulcher. Fischer et al. (2017b) showed that both chemical and visual predator cues are used independently by N. pulcher in predator detection, but they did not administer them together. We administered chemical cues in combination with visual cues and our results suggest that N. pulcher ignore cues from predator odour or conspecific skin extract in the presence of visual cues. Similar results were seen in juvenile roach, Rutilus rutilus, which responded differently towards visual and chemical predator cues, but when presented with both cues together, the response was similar to visual cues alone (Martin et al., 2010).

Artificial proxies can help adhere to the 3Rs (Reduce, Refine, Replace) of ethical framework (Russell & Burch, 1959). While this is also an excellent way to increase standardization, it is important to make sure that the employed proxies indeed have the desired effect. For example, a species or population living under consistently high predation risk may ignore predator cues unless immediate threat is sensed (Creel et al., 2008; Bell et al., 2009). In such a case, it is important use a proxy that can simulate an immediate threat to induce measurable response in the prey.

Conclusions

Here we show that N. pulcher do not use chemical cues from conspecific skin tissue as a indication of predation threat when combined with visual predator cues. The failure to respond to chemical cues may have been caused by precedence of visual cues over chemical cues when both cues in both modalities are presented together. We further show that including directed attacks by predator in a visual proxy induces responses similar to a live predator encounter. Most of previous work has focused on non-interactive predator visuals. We show that it is important to include prey-directed behaviour to simulate a threat level similar to that posed by a live predator.

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