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Abstract
ABSTRACT
Living in groups offers individuals a way of reducing their risk of predation. Visual lateralisation, characterised as an asymmetry in eye use, may offer an additional advantage to group‐living animals by enabling them to manage two concurrent visual tasks simultaneously. This could enhance multitasking efficiency by facilitating cohesion with group mates while monitoring for threats. In our study, we examined visual lateralisation of Trinidadian guppies (
Full text
Introduction
Predation is a major driver of group formation in prey species. Individuals in groups often experience higher survival compared to solitary individuals through mechanisms including risk dilution, the avoidance effect, the confusion effect and group vigilance (Ioannou 2021). Maintaining visual contact with other group members to sustain group cohesion while staying vigilant to threats is required to maximise an individual's chance of survival (Ward et al. 2011). However, performing two concurrent visual tasks may hinder the performance of both tasks unless visual information can be efficiently processed (Dadda and Bisazza 2006a; Dukas 2004; Rogers et al. 2013).
Within fish shoals, behavioural synchronisation among individuals is often high, particularly in environments with higher predation where shoaling is more prevalent and social tendencies of individuals (i.e., ‘sociability’) are heightened (Ioannou and Laskowski 2023a; Kelley and Magurran 2003; Seghers 1981). A complementary adaptation for individuals is a directional bias (left or right asymmetry) in certain behaviours, known as lateralisation or ‘handedness’ (Rogers et al. 2013). For example, some visual tasks may be preferentially performed with a certain eye (i.e., ‘visual lateralisation’), or an individual may demonstrate a consistent directional response (i.e., ‘motor lateralisation’). This is believed to be linked to a cerebral partitioning of cognitive functioning (Hulthén et al. 2021). Behavioural lateralisation is typically assessed through ‘relative laterality’, that is, the directional bias of the individual (left or right), and ‘absolute laterality’, the strength or intensity of this bias regardless of directionality (Brown et al. 2004; Bisazza and Brown 2011; Bisazza et al. 2000). Behavioural lateralisation has been observed across a range of taxa and behaviours including tool use, predator avoidance and escape responses (Rogers et al. 2013). More lateralised individuals have been demonstrated to have improved escape responses (shorter response latencies, higher turning rates and a longer distance travelled: Dadda et al. 2010), maintain foraging performance while being sexually harassed (Dadda and Bisazza 2006b), and tend to occupy safer positions in a group when under predator presence (Bibost and Brown 2013; Middlemiss et al. 2018) when compared to less lateralised individuals. However, despite the potential advantages of lateralisation, previous studies have not consistently observed it, with variability reported in both the directionality and strength of lateralisation across contexts, populations and species (Bisazza, Rogers, et al. 1998; Penry-Williams et al. 2022; Roche et al. 2020).
In environments with high levels of predation, individuals are expected to demonstrate enhanced visual lateralisation, an asymmetric bias in eye-use when viewing a stimulus. Fish species at lower trophic levels typically have laterally positioned eyes, offering limited binocular overlap (Vanegas and Ito 1983). This configuration means that stimuli are predominantly viewed by only one eye at a time (Middlemiss et al. 2018; Vanegas and Ito 1983). Lateralised eye-use (i.e., visual lateralisation) in fish has been extensively documented across various species and contexts, particularly in response to predatory (Brown et al. 2004; Facchin et al. 1999; Hulthén et al. 2021; Bisazza et al. 1997a; Broder and Angeloni 2014) and social (Fuss et al. 2019; Sovrano et al. 1999, 2001) stimuli. The information gathered from each eye is primarily processed by the contralateral hemisphere, allowing for the potential division of two concurrent visual tasks between brain hemispheres if one eye is used for each task (Bisazza and Brown 2011; Dadda et al. 2009). Cognitive partitioning, such as this, would enable more efficient information processing and multi-tasking (Bisazza and Brown 2011; Miletto Petrazzini et al. 2020; Vanegas and Ito 1983). Assuming that shoals are not so large and dense that most individuals can only see other shoal mates, which is relatively rare in freshwaters, this is particularly beneficial in the context of shoaling, as individuals could monitor shoal mates while simultaneously surveying other external stimuli, such as predators or food (Bisazza and Dadda 2005). Even for individuals in the middle of a shoal, their visual field is usually not so obstructed by shoal mates that they cannot see stimuli outside the shoal's perimeter (Strandburg-Peshkin et al. 2013). If specialisation of a visual task was dedicated to a particular eye, then detection latency and neural processing time would be minimised, allowing for an increased response efficiency (Bisazza et al. 2000; Brown et al. 2004; Dadda and Bisazza 2006a; Vallortigara, Rogers, et al. 1999). Furthermore, if multiple individuals within a shoal were to demonstrate lateralisation, this could enhance group synchronisation, cohesion and escape capacity (Brown 2005; Brown et al. 2004; Frasnelli and Vallortigara 2018; Miletto Petrazzini et al. 2020). In such circumstances, a mixture of individuals displaying right- and left alignment may be expected, dependent on their shoal positioning, as has been found in crimson-spotted rainbowfish (
However, most studies have focussed on assessing lateralisation in solitary individuals (Broder and Angeloni 2014; Facchin et al. 1999; Sovrano et al. 1999), despite many species, including guppies, often occurring in social groups. Furthermore, while some research has explored population-level lateralisation in dichotomous ‘high versus low’ predation environments (Brown et al. 2004), few studies have directly examined how the presence of a live predator influences lateralisation in real-time, particularly in a social context. The gap in our understanding is significant, as the benefits of lateralisation are theorised to be especially pronounced in group-living species facing predation pressure (Brown et al. 2004).
Recent work has begun to address some of these limitations. Johnson et al. (2020), working with Xenophallus umbratils, included a predator as a stimulus in their arena trials and observed a strong impact on lateralisation. However,
The first aim of this study was to validate that the guppies perceived the predatory stimulus as a threat. This was assessed by measuring the guppies' ‘attack cone avoidance’ around the acara; that is, the guppies should avoid being directly in front of the acara's head if they perceive this stimulus as a potential threat (Magurran and Seghers 1990). Next, the second aim was to determine whether shoaling and/or a live predatory stimulus impacts visual lateralisation. We hypothesised that both maintaining shoaling behaviours in a group and viewing a live predator should enhance lateralisation, with a potential additive effect when multi-tasking both activities. The final aim was to assess the repeatability of visual lateralisation (Penry-Williams et al. 2022; Vinogradov et al. 2021). We hypothesised that if visual lateralisation is an adaptive anti-predatory trait, then this behaviour should be more consistent and repeatable when fish are exposed to the predatory stimulus than when they are not (Toscano et al. 2014).
Materials and Methods
Study Species: Trinidadian Guppies (
Trinidadian guppies have been previously used as a model organism for investigating the effect of predation risk on lateralisation (Broder and Angeloni 2014; De Santi et al. 2000; Irving and Brown 2013). The fish used in this investigation were descendants of wild guppies collected from a high-predation environment in the Guanapo River in Trinidad (Moonan: 10.6082° N 61.2547° W) in April 2019 by the Guppy Project (University of Oxford). Guppies were exported to the John Krebs Field Station (Oxford, UK) where they were selectively bred across three generations to prevent inbreeding and maintain genetic diversity. Guppies were maintained between 25°C and 27°C and fed twice daily with either live brine shrimp nauplii or liver paste, with a 12:12 light:dark photoperiod. Guppies were transferred to the University of Bristol (Bristol, UK) in December 2020 by car for approximately 1.5 h. All fish were alive and in good condition upon arrival at the University of Bristol. Preceding the investigation, guppies were maintained in mixed-sex groupings of approximately 50–100 individuals in 90 L holding tanks (length × width × height: 70 × 40 × 35 cm) furnished with plastic foliage and a sand substrate. Guppies were fed once per day ad libitum with either brine shrimp or fish flake and maintained at 26°C–28°C and a 12:12 light:dark photoperiod. Guppies used in this investigation had previously been tested in a study exploring the short-term effects of temperature and turbidity on social behaviours in February and March 2021 (detailed in Allibhai et al. 2023), at least 2 months prior to use in the current study. Despite descending from individuals from a high-predation habitat, the individuals used were naïve to predators before being used in the current study.
Only female guppies were used for this experiment to standardise the experimental protocol due to their stable social interactions (Croft et al. 2006) and to exclude sexual behaviours and harassment from males (Cummings 2018). Sexual segregation in guppies does occur in the wild (Croft et al. 2006); thus, testing all-female groups is ecologically relevant. Female guppies were sorted into size-selected groups (ngrp = 32) to recognise individuals across trials without the need for elastomer tagging. Being able to recognise individuals across trials for the same group was desirable to directly compare each individual's behaviour across the different treatments and avoid pseudoreplication by being able to include individual identity as a random effect in the models. This also allowed us to test for repeatability at the individual, as well as group, level (in contrast, see the study by Clark et al. (2025) where individual guppies could not be identified over multiple trials with the same group). Guppies were measured (mean standard length ± SD: 26.1 ± 5.7 mm) and sorted into size classes: small (< 22 mm), medium (23–29 mm) and large (> 30 mm). One individual from each size class was haphazardly selected to form each group of three fish (mean group length ± SD: 26 ± 1 mm); guppies are often found in small groups in the wild with variation in body size between individuals (Clément et al. 2017). Small deviations from size classifications were made in a few instances where fish in a particular size class had limited availability (nind = 6). Groups were viewed on the camcorder used for the trials to ensure individuals could be recognised and identified within their groups. Groups were housed in separate breeding nets (length × width × height: 16 × 13 × 13 cm) for the duration of the experimental period (18 days) to maintain group identities (ngrp = 32) with two breeding nets per 45 L holding tank (length × width × height: 35 × 40 × 35 cm). Breeding nets were kept in tanks on a filter system to ensure adequate water quality, oxygenation and to prevent temperature fluctuations. Groups were acclimatised for 72 h prior to testing. In instances where an individual within a group died during the experiment, they were replaced with a size-matched individual (nind = 12). The new individual and group were assigned a new individual ID and group ID, respectively and acclimatised for 24 h prior to testing.
During the investigation, all water quality parameters (pH, ammonia, nitrates and nitrites) were within the recommended range for the species and were monitored on the recirculated filter system weekly. Guppies were fed commercial fish pellets at 17:00, approximately 16–24 h before trials.
Study Species: Blue Acaras (
Blue acaras are a major predator species of Trinidadian guppies (Deacon et al. 2018; Zanghi et al. 2024). A total of 14 blue acaras were used as the predatory stimulus to induce a heightened perception of predation risk (mean standard length ± SD: 86 ± 11 mm). The blue acaras could not be sexed reliably, so we could not factor this into the analyses. These were bred at the University of Bristol from stock provided by the University of Exeter and were individuals used in an earlier study of their prey pursuit behaviour (Szopa-Comley and Ioannou 2022, 2025), demonstrating their predatory tendencies. Acaras were held in a 90 L holding tank (length × width × height: 70 × 40 × 35 cm) furnished with plastic foliage, plastic tubes and a sand substrate base. Acaras were fed commercial fish pellet at 17:00, approximately 16–24 h before trials. Acaras were maintained at a temperature of 26°C–28°C and a 12:12 light:dark photoperiod. Individual acaras were used in a maximum of one trial each day to limit potential stress.
Lateralisation Assays
The apparatus used in this experiment was similar to that used by Broder and Angeloni (2014) and Zanghi et al. (2023). The assay consisted of a white circular container (Figure 1a,b: diameter × height: 32 × 30 cm) with a central transparent chamber containing either a blue acara (Predator treatment) or left empty (Control treatment). The predators were restricted to the central compartment to avoid olfactory cues from the predator and prevent direct physical contact between predator and prey. Groups of guppies or a solitary guppy were introduced into an opaque white acclimatisation section (white PVC tube: diameter × height: 5.5 × 10 cm) in the area surrounding the central chamber for 10 min preceding the trial, after which the video camera was started (Panasonic HC-X920: 1920 × 1080 pixel resolution, 50 frames/s) and the acclimatisation tube was lifted. Trials lasted for 15 min. All tanks were shaded from direct light using a translucent plastic sheet to prevent a light-induced turning bias and to reduce reflections on the surface of the water to facilitate computer tracking from video. Water temperature was between 26°C and 28°C and was replaced from the filtration system following each trial. Water level was maintained at 8 cm in the outer ring and 11 cm within the central chamber.
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Trials took place in two testing blocks in June and July 2021. Treatments manipulated both whether the predator was present or absent and whether guppies were tested alone (nind = 96) or in their group (ngrp = 32) in a fully factorial design; the treatments were ‘Predator-Solitary’, ‘Predator-Group’, ‘Control-Solitary’ and ‘Control-Group’. Each individual was subject to all four treatments twice (i.e., eight trials total; Figure 1c), with the treatment order following a Latin-square design; each individual was tested once every 2 days. Two trials were run in parallel, with a ‘Predator’ trial and a ‘Control’ trial taking place concurrently. ‘Group’ and ‘Solitary’ treatments took place on alternating days of testing for each group. Control and Predator treatments were alternated between the two experimental setups after assaying each group to account for potential asymmetries; whether the trial took place in the left or right setup was included in the statistical analyses. The testing order of fish groups was randomised each day using . In the Solitary treatment, the testing order of individuals within each group was also randomised. Solitary trials occurred between 9:00 and 17:00 (n = 24 trials per day). To accommodate for the fewer trials on days that Group trials took place (n = 8 trials per day), start times were varied. Group trial start times were randomly determined each day to fall within the windows of 9:00–11:00, 11:00–13:00 or 13:00–15:00.
Data Processing: Guppy Trajectories
All trial videos were converted from .MTS format to .mp4 (1920 × 1080 pixel resolution, 30 frames/s) using Handbrake (v1.2.0) (The Handbrake Team 2018) to facilitate tracking in idTracker (v2.1) (Pérez-Escudero et al. 2014). Following tracking, trajectories were manually assessed for accurate identification of guppies and their trajectories. Where inconsistencies were found, tracking parameters were adjusted and tracking was reperformed. The centre and diameter of the central chamber were manually measured in ImageJ (v1.52a) (Rasband 2018) from still images of each trial obtained using VLC media player (v3.0.8 Vetinari) (VideoLan 2006). Data cleaning involved the removal of datapoints exceeding maximum speeds for Trinidadian guppies (140 cm/s burst speed) to account for potential tracking errors (Chappell and Odell 2004; Oufiero and Garland 2009). Data were then interpolated using the na.interpolation function (imputeTS package) (Moritz and Bartz-Beielstein 2017) using a linear option and allowing a maximum gap of 10 successional missing points (approximately 0.33 s) in R (v3.6.1) (R Core Team 2019) with RStudio (v1.2.1335) (RStudio Team 2019).
For each frame of video (30 frames/s for 15-min videos), relative lateralisation was calculated using χ = arcsin (sin (θ − ϑ)), in which θ was the angle of the fish between two frames and ϑ is the angle of the arena radius through the position of the fish (Herbert-Read et al. 2015; Penry-Williams et al. 2022). Lateralisation indexes are relative to the centre of the central chamber. A score of χ > 0 demonstrates a clockwise orientation, that is, using the right eye to view the centre of the arena, while a score of χ < 0 is an anti-clockwise orientation, that is, using the left eye to view the centre of the arena. While the number of frames demonstrating right and left-eye use was combined in the statistical analysis to assess relative lateralisation as a binomial variable, to display the data, these were used to calculate a relative laterality (RL) index (on a scale of −1 to 1) using RL = ((Nright − Nleft)/(Nright + Nleft)), where Nright is the number of frames of right-eye use and Nleft is the number of frames of left-eye use. To calculate absolute laterality (AL) to assess the intensity of lateralisation, the absolute values of the relative laterality index were calculated, so that absolute laterality ranged from 0 to 1. Each individual's activity was calculated as their speed (cm/min) from the total distance travelled by the fish (cm) and divided by the trial duration (min).
Data Processing: Acara Orientation and Attack Cone Avoidance
An assumption of our study is that the guppies perceive the blue acara as a predatory threat, although this needed to be verified, especially with using predator-naïve fish. Therefore, in trials with a predator, whether the guppies avoided the attack cone of the acara was assessed, that is, whether the guppies avoided being directly in front of the acara's head (Magurran and Seghers 1990). This was measured by assessing the guppies' position in relation to the acara's body orientation.
An automated custom ImageJ plugin using a convolution-based approach was used to detect the centre of mass and orientation of the acara (details available in Heathcote et al. 2020). The extracted angle and centre of mass were used to calculate the relative positioning of each guppy for each extracted frame of the trial, with 0° being directly in front of the acara's head and 180° behind the acara's tail. The activity of the acara was also assessed through the change in degrees per minute.
To facilitate tracking, the resolution and frame rate of the trial videos were reduced using ffmpeg (640 × 360 pixel resolution, 3 frames/s) (FFmpeg Developers 2021). This reduction for evaluating the orientation of the acara did not impact the results. To verify the accuracy of the tracking, a subset of the trial videos (n = 10) was randomly selected and run at the full resolution (1920 × 1080 pixels resolution) and full frame rate (30 frames/s). The angles extracted from the full resolution and frame rate videos were found to be in close agreement with those from their downsampled version. The mean ± SD absolute difference in degrees was 0.10° ± 0.31° for resolution and 0.70° ± 0.67° for frame rate. Further comparisons of the guppies' positioning relative to the acara's head also showed good agreement between the full and downsampled videos. The relative difference between the output datasets, calculated as (full frame rate/reduced frame rate) × 100, had a mean ± SD of 99.75% ± 0.49% for resolution and 100.03% ± 4.06% for frame rate, indicating a high level of consistency between the full and downsampled datasets.
Data Analysis
All statistical analysis was performed in R (v3.6.1) (R Core Team 2019) with RStudio (v1.2.1335) (RStudio Team 2019). All analyses were conducted on a per-individual, per-trial basis, that is, with a single line of data for each trial of solitary fish, and three lines of data for each group trial (one per-individual in the group). Summary statistics (e.g., medians and proportions) across each trial for each individual were calculated from the tracking data to avoid pseudoreplication; the remaining non-independence in the data was accounted for by the inclusion of random effects. In group trials, the data across the three individuals in the group were not averaged. To validate guppies perceiving the blue acara as a predatory threat, it was initially assessed whether there was attack cone avoidance around the head of the acara, in trials with a predator present (Magurran and Seghers 1990). Individual guppy positioning relative to the acara (median degrees from the acara's head) was analysed as a response variable using a linear mixed-effects model (LMM). Explanatory variables were grouping treatment (‘solitary’ or ‘group’), guppy size (mm), guppy activity (cm/min), replicate number (first or second time completing that treatment), time of day, date, trial number (one to eight), side of assay (left or right setup), testing block (first or second set of groups), predator activity (degrees turned/min) and predator size (mm), with individual ID nested within-group ID as the random effect.
Visual lateralisation was then investigated across all trials. Absolute laterality indexes (AL) were rescaled from 0–1 to 0–100 and rounded to the nearest whole number to fulfil the assumptions for a negative binomial generalised linear mixed-effects model (GLMM) using the glmer.nb function (lme4 package) (Bates et al. 2015); a negative binomial error distribution was necessary due to the strong right (positive) skew in the distribution of absolute laterality. Relative lateralisation was assessed by using the proportion of frames with left versus right eye use in a binomial GLMM using the glmer function (lme4 package). To fulfil model assumptions, the proportion of right versus left-eye use over each trial (27,000 frames) was scaled to 100. Grouping treatment (‘solitary’ or ‘group’), predation treatment (‘predator’ or ‘control’), guppy size (mm), guppy activity (cm/min), replicate number (first or second), time of day, date, trial number (1–8), side of assay (left or right setup) and testing block (first or second) were included as main effects, with individual ID nested within-group ID as the random effect. For all models, the main effect with the highest p value (when > 0.05) was removed from each iteration of the model (i.e., backward selection), and the model was re-run until only significant main effects remained. Grouping treatment and predation treatment were initially included as an interaction term; however, this interaction was removed if it had the highest p value (> 0.05), which was the case in all models. Grouping treatment and predator treatment were not removed from the models as explanatory terms at any stage, as these are integral to the hypotheses being tested.
Data from trials with a predator were also analysed separately to assess whether additional factors significantly impacted guppy behaviour. In these models, reflecting those previously described, predator activity (degrees turned/min), guppy positioning relative to the acara (median degrees from the orientation of the acara's head), and predator size (mm) were added to the models as main effects along with the aforementioned main and random effects, excluding the predator treatment explanatory variable. The assumptions of all models were verified with QQ plots and residuals versus fitted values using the residual diagnostics for hierarchical (multi-level/mixed) regression models (DHARMa) package (Hartig 2019). Variance inflation (multicollinearity) was assessed using the vif function (car package) (Fox and Weisberg 2011). Individuals not completing a minimum of one of each of the treatments were excluded from the analysis (Nind = 12, Nobs = 15).
Repeatability (R) in guppies' behavioural traits (positioning relative to the acaras' head, absolute laterality and relative laterality) was assessed using repeatability estimates with parametric bootstrapping at 1000 iterations (rptR package) (Stoffel et al. 2017). This was initially run on all trials together and then on subdivisions of each treatment (‘Predator-Solitary’, ‘Predator-Group’, ‘Control-Solitary’ and ‘Control-Group’). Median degrees from the acara's head were assessed with a Gaussian datatype option. For absolute lateralisation, individuals were ranked (with averaged tied ranks) to avoid the violation of homogeneity of variances in the model's residuals, and an estimate of repeatability was assessed via a Gaussian datatype option. Relative lateralisation was assessed through the proportion of frames with left versus right eye use (left and right) using the proportion datatype option. For all models, repeatability in both individual ID and group ID was investigated. LMMs and GLMMs with the same model structure were fitted to check and verify the model assumptions.
Ethical Note
All experimental procedures and housing conditions were approved by the University of Bristol Animal Welfare and Ethical Review Body (UIN/21/003). All fish were monitored during the experimental period to ensure that they did not display overt signs of stress, and after testing were retained in the laboratory for use in future experiments.
Results
Attack Cone Avoidance
The guppies displayed attack cone avoidance (measured by median degree from the acara's head), positioning themselves at 123° ± 10° (mean ± SD) for the solitary trials and 125° ± 7° for the group trials (Figure 2). Guppies spent 73.18% ± 7.50% (mean ± SD) and 71.42% ± 5.60% of the trial behind the predator (> 90°) for the solitary and group trials, respectively. No explanatory variable significantly affected the guppies' attack cone avoidance, including whether the guppies were tested alone or in groups (Figure 2: Grouping treatment: LMM: χ2(1) = 1.94, p = 0.163).
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Across the treatments, repeatability in attack cone avoidance (measured by median degrees from the acara's head) was not found among individuals (LMM: R = 0.023, 95% CI = 0–0.113, Nobs = 379, Nind = 97, p = 0.301), nor among their respective groups (R = 0.034, 95% CI = 0–0.29, Nobs = 379, Ngrp = 38, p = 0.132). However, when analysing the treatments separately, group ID did produce significantly repeatable attack cone avoidance behaviour within the ‘Predator-Group’ treatment (Table 1), as well as individual ID for the ‘Predator-Solitary’ treatment. Group ID was not found to be repeatable in the solitary trials, nor individual ID in the group trials (Table 1).
TABLE 1 Repeatability of behavioural parameters (attack cone avoidance, absolute laterality and relative laterality) of female
| Treatment | Individual ID | Group ID | ||||
| Repeatability (SE) | 95% CI | p | Repeatability (SE) | 95% CI | p | |
| Attack cone avoidance | ||||||
| Predation-Group | 0.000 (0.045) | 0.000–0.147 | 1.000 | 0.381 (0.087) | 0.196–0.522 | < 0.001 |
| Predation-Solitary | 0.167 (0.094) | 0.000–0.324 | 0.019 | 0.000 (0.038) | 0.000–0.124 | 1.000 |
| Absolute laterality | ||||||
| Predation-Group | 0.000 (0.061) | 0.000–0.203 | 1.000 | 0.129 (0.070) | 0.000–0.265 | 0.020 |
| Predation-Solitary | 0.123 (0.089) | 0.000–0.301 | 0.107 | 0.000 (0.033) | 0.000–0.109 | 1.000 |
| Control-Group | 0.000 (0.054) | 0.000–0.182 | 0.500 | 0.287 (0.083) | 0.117–0.441 | < 0.001 |
| Control-Solitary | 0.028 (0.065) | 0.000–0.222 | 0.395 | 0.000 (0.030) | 0.000–0.106 | 1.000 |
| Relative laterality | ||||||
| Predation-Group | 0.000 (0.000) | 0.000–0.002 | 1.000 | 0.004 (0.001) | 0.001–0.007 | < 0.001 |
| Predation-Solitary | 0.003 (0.002) | 0.000–0.008 | 0.157 | 0.000 (0.001) | 0.000–0.003 | 1.000 |
| Control-Group | 0.000 (0.000) | 0.000–0.002 | 1.000 | 0.008 (0.002) | 0.003–0.013 | < 0.001 |
| Control-Solitary | 0.010 (0.004) | 0.002–0.017 | 0.004 | 0 (0.001) | 0.000–0.005 | 0.427 |
Absolute Lateralisation
Low levels of absolute lateralisation were found across all treatments. A large proportion of the trials, 98.94%, were below an absolute laterality index of 0.5, with 58.38% below 0.1. Despite the low levels, several factors were found to have a statistically significant impact on absolute lateralisation. Solitary trials produced significantly higher absolute lateralisation indexes (mean absolute laterality index ± SD: 0.13 ± 0.12) compared to group trials (0.09 ± 0.09: Figure 3: GLMM: χ2(1) = 51.79, p < 0.001). Control trials demonstrated higher absolute lateralisation indexes (0.12 ± 0.11) compared to predation trials (Figure 3: 0.10 ± 0.10: χ2(1) = 5.06, p = 0.024). Guppy activity (cm/min) was positively correlated with absolute laterality index (χ2(1) = 32.74, p < 0.001). Analysing the predation trials with the inclusion of predator-specific parameters in the model did not qualitatively alter these results, with both the grouping treatment (χ2(1) = 28.90, p < 0.001) and guppy activity (χ2(1) = 23.62, p < 0.001) being the only explanatory variables significantly impacting absolute laterality.
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Repeatable absolute laterality indexes were observed for the group ID random effect when data from all treatments were included in the analysis (LMM: R = 0.050, 95% CI = 0–0.112, Nobs = 752, Ngrp = 44, p = 0.012); however, individual ID was only marginally repeatable (R = 0.039, 95% CI = 0–0.099, Nobs = 752, Nind = 99, p = 0.052). When assessing treatments separately, group ID was significantly repeatable within both predation and control group trials (Table 1) but not within the solitary trials. Absolute lateralisation for individual ID was not repeatable in any of the treatments (Table 1: p = 0.107–1).
Relative Lateralisation
Low levels of relative lateralisation were identified in all treatments. However, there was a significant leftward bias in viewing the central chamber when the predator was present (mean relative lateralisation index: −0.01 ± 0.14) compared to the control trials (Figure 4: 0.01 ± 0.16, GLMM: χ2(1) = 4.22, p = 0.040). Trials later in the series also had a significant leftward trend (χ2(1) = 4.01, p = 0.045). Relative lateralisation was not different between the solitary and group treatments (Figure 4: χ2(1) = 0.09, p = 0.760). Analysis of the predation treatment including the additional predator-specific parameters bore no significant predictors of relative lateralisation (Grouping treatment: χ2(1) = 2.25, p = 0.133).
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An individual's relative laterality was repeatable when including all treatments (GLMM: R = 0.003, 95% CI = 0.001–0.005, Nobs = 752, Nind = 99, p < 0.001), as well as within each group (R = 0.002, 95% CI = 0–0.003, Nobs = 752, Ngrp = 44, p = 0.019). Investigating the repeatability of relative lateralisation in each treatment separately identified, again, that group ID was significantly repeatable within the predation and control group treatments (Table 1), and this was not maintained in the solitary treatments (Table 1). Individual ID was significantly repeatable within the ‘Control-Solitary’ treatment (Table 1) but was not repeatable within any of the other treatments (Table 1).
Discussion
Effect of Predation Risk on Lateralisation
Previous studies have found that visual lateralisation is enhanced in response to a predatory threat (Bisazza et al. 1997a; Bisazza, Facchin, et al. 1998) or when shoaling (Brown et al. 2007; Dadda et al. 2012; Sovrano et al. 1999). There are benefits expected in being able to observe both predators and shoal mates simultaneously (Dadda and Bisazza 2006a; Rogers et al. 2013). Being able to monitor conspecifics while simultaneously observing predators would minimise detection latency and neural processing time, maximising response efficiency and group synchronisation (Bisazza et al. 2000; Brown et al. 2004; Vallortigara, Regolin, et al. 1999). Even though the guppies used were predator naïve, it is evident they perceived the blue acara as a predatory threat, with a clear avoidance of the predator's attack cone (Magurran and Seghers 1990), even though blue acaras tend to orient themselves so that their prey are directly ahead, within their binocular field of vision (Szopa-Comley and Ioannou 2025). This evidence indicates that the guppies were visually engaged and responding to the predator's movements based on visual stimuli, rather than exhibiting responses attributable solely to motor lateralisation. Despite this, absolute visual lateralisation was not enhanced when viewing the predator nor when shoaling with a group. Notably, absolute lateralisation was also not enhanced when simultaneously performing these activities as there was no significant interaction between these two treatments. However, we did find a statistically significant, albeit small, preference for left-eye use when facing a predator in the central chamber across both group and solitary trials.
In our study, we observed low levels of absolute lateralisation across all experimental conditions. Control trials, however, showed higher absolute laterality than trials containing the live predator, contrary to predictions based on previous studies (Brown et al. 2004; Broder and Angeloni 2014; Facchin et al. 1999; Sovrano et al. 1999). This result runs counter to the expected trend, where predator presence is typically associated with increased lateralisation, though it aligns with a less common finding as reported in adult fathead minnows (
Despite the lower absolute lateralisation found in our predator trials, guppies did demonstrate a significant leftward bias compared to control trials. Information from the left eye is primarily processed by the contralateral right hemisphere of the brain. In fish, the right hemisphere is primarily associated with vigilance, fear responses and predator detection (Cantalupo et al. 1995; Facchin et al. 1999; Sovrano et al. 2001; Vallortigara and Rogers 2005), while the left hemisphere is linked to routine behaviours like feeding and social interactions (Bisazza et al. 1997b; Miklosi and Andrew 1999; Rogers et al. 2013). Previous research has similarly identified a trend in right eye use (left turning) in goldbelly topminnows (
Effect of Being in a Group on Lateralisation
Absolute lateralisation was found to be significantly higher when fish were tested alone compared to when they were tested in groups, contrary to our prediction. As a shoaling species, solitary individuals may experience a higher level of perceived threat, stress or fear when alone, regardless of there being a predator present. Stress has been found to be a key factor in determining the degree of behavioural lateralisation, with higher levels of stress contributing to higher levels of behavioural lateralisation (Berlinghieri et al. 2021; Halpern 2005; Ocklenburg et al. 2016). It is possible that solitary individuals would make better use of visual lateralisation to monitor their surrounding environment for predators and complete other tasks, such as foraging, while groups may gain sufficient protection from collective vigilance (Ward et al. 2011). However, despite the importance of the social context for lateralisation, most studies have only assessed solitary individuals (Brown et al. 2004).
In our study, a significant interaction between the grouping treatment and predator presence was not identified. However, previous work has identified relationships in visual lateralisation for anti-predatory and social contexts. Despite no population-level asymmetric eye-use, in two separate assays goldbelly topminnows (
The Repeatability of Lateralisation
Previous assessments have found mixed results regarding the repeatability of lateralisation in solitary individuals (Penry-Williams et al. 2022; Roche et al. 2020; Vinogradov et al. 2021). Roche et al. (2020) found that relative lateralisation in five fish species was not repeatable in a detour assay, including in feral guppies (
A number of studies have demonstrated consistent among-group behavioural variation in fish shoals, also known as group personality variation, including in guppies (Clark et al. 2025). This can occur even when groups are formed of randomly selected individuals (Jolles et al. 2018) or group membership is designed to minimise inter-group variation (MacGregor and Ioannou 2021). Consistent differences among groups can arise from consistent inter-individual variation among individuals, where individual differences in behaviour persist in groups; this was unlikely to have been a factor in our study as repeatability at the level of the individuals was limited, and the importance of the group identity random effect was only important in group trials. Instead, these results suggest social conformity, where individuals adjust their behaviour to match the behaviour of their group mates (Ioannou and Laskowski 2023b). In animal groups, conformity is necessary for collective movement and consensus decision making (Wade et al. 2020), and the tendency for fish shoals to align their orientation in the same direction (as in Figure 1b), known as polarisation (Ioannou and Laskowski 2023a), allows all individuals to use the same eye to view the same stimulus. However, it is not clear why, either functionally or mechanistically, the groups varied consistently in their absolute and relative lateralisation over multiple trial days apart in our study; this deserves further investigation.
The manipulation of shoal compositions based on body size to allow for individual identification in our study may have had an impact on the results regarding repeatability. Forming shoals of individuals that are more diverse in body size should have increased within-group variation in behaviour relative to among-group variation. This should have reduced the differences among groups, and hence reduced the repeatability in the group identity random effect (Ioannou and Laskowski 2023b). Thus, the strength of social conformity in lateralisation may be underestimated, and social conformity may be playing a major role in determining eye-use in social species. Consistent with this, in a study of 16 fish species, shoaling species (e.g., Poeciliidae and Cyprinidae) were more likely to demonstrate a population-level conformity in the direction of lateralisation when undertaking predator escape responses, while non-shoaling species (e.g., freshwater Gobiidae and Ancistrus sp.) were more likely to demonstrate a mixture of individuals with both right and left alignment (Bisazza et al. 2000).
In our study, it was hypothesised that if lateralisation is an important anti-predation mechanism, then the presence of a predator should reduce the variability between individuals in this trait in order to maximise survival under perceived predation threat (Toscano et al. 2014). In agreement with this hypothesis, repeatability in relative lateralisation was identified in the solitary trials at the level of the individual in the control trials, but not in the trials with a predator stimulus. Alternative anti-predation mechanisms may also be adopted by solitary individuals in the predator trials, such as ‘protean’ movements, which may lead to unpredictable swimming behaviour and, therefore, measured eye-use (Jones et al. 2011; Szopa-Comley and Ioannou 2022). Not mutually exclusively, the movement of the guppies in the predator trials could have been responding more to avoiding the attack cone of the predator, rather than their movement being primarily to determine with which eye to observe the predator. Behaviours more sensitive to environmental or motivational influences may be less predictable and repeatable (Bell et al. 2009), such as those influenced by energetic needs (MacGregor et al. 2021), social interactions (MacGregor and Ioannou 2021; Rands and Ioannou 2023), or ecological variables (Castellano et al. 2002; Roy and Bhat 2018; Smith and Hunter 2005).
Conclusions
This investigation provides evidence suggesting that visual lateralisation is not enhanced under predation threat or in social groups, contrary to our predictions, with relatively low lateralisation indexes observed across all treatments. Repeatability of lateralisation indexes with the group-level random effect was identified, but limited repeatability at the individual level suggests an important role of social conformity in lateralisation. We suggest that these results indicate social processes may have a greater effect than predation risk on variation in lateralisation. Given the predictions of lateralisation generally suggesting greater benefits to groups over solitary individuals, we suggest that a greater consideration of groups when assessing lateralisation, in addition to individuals, is required to disentangle the potential benefits of visual lateralisation.
Author Contributions
Iestyn L. Penry-Williams: conceptualization (lead), data curation (lead), formal analysis (lead), investigation (lead), methodology (lead), project administration (lead), visualization (lead), writing – original draft (lead). Culum Brown: funding acquisition (supporting), supervision (supporting), writing – review and editing (lead). Christos C. Ioannou: conceptualization (supporting), funding acquisition (lead), methodology (supporting), project administration (supporting), resources (lead), supervision (lead), writing – review and editing (supporting).
Acknowledgements
This research was supported through the NERC (Natural Environment Research Council) GW4+ Doctoral Training Partnership and Macquarie University Cotutelle (NE/L002434/1). We would like to thank Tomos Potter, Anja Felmy and the Guppy Project at the University of Oxford for the collection, breeding and transport of guppies used within this research. We would also like to thank Robert Heathcote, Jolyon Troscianko and collaborators for access to their custom ImageJ plugin for assessing fish orientation, originally presented in Heathcote et al. (2020). We also thank Anne-Kristin Lenz for helpful discussion on extraction protocols and calculations.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data for the analyses are provided as Supporting Information S1.
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