Study subjects

Seven captive-bred leopard geckos were used in the study. The subjects comprised five females and two males, whose ages ranged from 2 (juvenile) to 14 years (Table 2). Subjects here were kept in five heated vivaria (constant temperature of 28 ± 2 °C), housed in duos (LB and SK; DO and JY) or singly (LO, Un-named 1, Un-named 2) at the study location, Broomfield College Animal Care Centre, Derby College, U.K. DO and JY had been kept together for 2 years prior to the onset of the study. LB and SK were paired temporarily for the study period. The three singly housed individuals had also been kept singly prior to the study period. Subjects had no previous exposure to individuals other than where stated above.

Subjects were fed twice daily at 07.00 and 13.00. Morning trials were conducted post 9 am and afternoon ones post 2.30 p.m. Since leopard geckos are crepuscular, we conducted our trials with the lights turned off in the experiment room, relying only on lighting from those vivaria present. All of our subjects were experimentally naïve.

Apparatus

Experimental arenas comprised two glass-sided fish tanks (35 × 25 × 24 cm), positioned horizontally and placed end to end (Fig. 1). The adjoining ends allowed clear visibility into the other tank from ground level to 6 cm high, sufficient to easily view a conspecific through. A wooden occluder (25.5 × 19.7 cm) was set in an angled positioned between the two tanks. The lids of the tanks were made of black plastic comprising a transparent, rectangular, plastic flap that could be lifted to reach in to access the subjects. The lid and the flap were kept closed during the trials. To reduce the possibility of laser light reflection onto the flap, the underside of the flap was covered with newspaper. The three sides of the demonstrator’s tank were also covered with newspaper to reduce any reflected laser light against the sides of the tank. A StreamLight Stylus Pro® Penlight (model no. 66124) with a green laser light was shone onto the occluder from outside the tank, behind on the demonstrator’s side. Therefore, the demonstrator, but not the observer, was able to see and orientate toward the laser light. The laser was handheld by the experimenter, so that the laser could be projected freely across horizontal occluder rather than just being shone in a fixed position. Switching the laser pen on/off appeared to not generate any audible interference.

Fig. 1 The experimental set up. Tanks were set end to end allowing ground-level visual access to the demonstrator (d) by the observer (o). Access to where the demonstrator looked (site of laser projection) was blocked, for the observer, by the occluder. During the trials, the lids of the tanks were fastened Full size image

Pre-testing

Pre-testing trials were undertaken to select a demonstrator for the main study. Each subject was tested in five trials over a 2-day period (July 2017). Because of the potential for male aggression toward unfamiliar individuals (Mason and Gutzke 1990), we tested only our five females for the role of demonstrator. We used the criterion of most “look ups” to select the testing phase demonstrator. Adult female (LB) was chosen, since she reliably looked up (head and neck orientation) toward the laser stimulus. The remaining six subjects were therefore assigned the role of observer.

Looking up behaviour

We recorded the looking up behaviour performed by the demonstrator and the following response by the observer to the demonstrator looking up at the laser. When defining looking up behaviour in leopard geckos, our pre-testing observations established the presence of three different forms of “looking up” behaviour (Table 1).

Table 1 Definitions of “looking up” behaviour in leopard geckos Full size table

Procedure

The main testing phase of the study was conducted over a 3-week period (July–Aug. 2017) and we limited trials to Mondays, Wednesdays and Fridays to reduce the potential for habituation that daily testing might have induced. Subjects were each exposed to three testing periods: the experimental condition and two controls. Each trial condition lasted 60 s. We randomised presentation order to reduce the possibility of order effects. All subjects received the same number of trials with each of the six given three trials of the corresponding conditions (i.e. 3 × 3 trials each).

Experimental condition

In the experimental condition, a gaze-following response was recorded if the observer extended their head and neck toward the stimulus or climbed the tank and then extended their neck and head toward the stimulus (see e.g., Loretto et al. 2010; Wilkinson et al. 2010; Table 1). A “look up” response was scored for any look up with a minimum duration of 10 s. Since the laser was being shone onto the top section of the tank above the angled occluder, to attempt to orientate toward where the demonstrator was looking, the observer’s looking up action was unambiguous.

No laser control

In this trial, to rule out the possibility the observer was simply looking up when faced by a conspecific, no laser was projected and the demonstrator, although present, was not encouraged to look up (no light beam). We terminated (and re-ran) the trial if the demonstrator did not refrain from looking upward for the full 60 s. For any of the trials that were prematurely terminated, these trials were not included.

No demonstrator control

In the no demonstrator control condition, the demonstrator was removed to test for the possibility that the observer was able to see the laser pointer and the light stimulus, rather than it being the demonstrator’s actions, that was cueing their looking response. The condition was identical to the experimental condition except with no demonstrator present. The observer was kept still in their tank facing the adjoining tank, while a laser was shone onto the occluder. A look up response could imply the stimulus was visible to them. A null response was recorded if the observer failed to look up.

Statistical analysis and inter-rater reliability

We used a repeated-measures study design based on the combined number of “look ups” exhibited by each individual across all three trials. Due to a limiting sample size, we applied a Friedman’s non-parametric test to detect the difference in look up behaviour under different test conditions across each of the individuals. We employed Bonferroni-corrected Wilcoxon signed-rank tests for post hoc testing. The effect size was calculated using r = z/√N. According to Cohen (1988), the effect size threshold of r = 0.1 is small; r = 0.3 is medium and r = 0.5 is large. The statistical tests were conducted using IBM SPSS (version 24.1).

To validate our coding procedure, we ran a separate series of trials over 1 day in August 2017. Those trials were for used for inter-observer reliability purposes only and are not included in the data set presented. Trials followed the same procedure of the experimental condition and the two control conditions. Testing was filmed using an Apple iPad 2 fixed to a stand at the back of the observer’s tank, facing the adjoining tank. Videos were labelled according to trial type. An independent coder (blind to experimental condition) was trained to recognise ‘following’ responses using still images showing the different sorts of responses used by leopard geckos when gaze following. These included head and neck orientation toward the occluder and attempting to climb the front of the testing tank coupled with head and neck alignment toward the light stimulus (Table 1). The primary coder (JAS) coded 100% of the 31 videos. The independent coder then coded 16 randomly selected videos. Inter-rater reliability testing was performed to determine the level of uniformity between the two coders. Inter-rater reliability was high with 80% agreement in the responses recorded. Applying Cohen’s k to determine the level to which this agreement could be attributed to chance highlighted a moderate to good level of agreement between the two coders k = 0.57, p < 0.0001.