Experimental arena settings might lead to misinterpretation of movement properties

Movement is an important animal behavior contributing to reproduction and survival. Animal movement is often examined in arenas or enclosures under laboratory conditions. We used the red flour beetle (Tribolium castaneum) to examine here the effect of the arena size, shape, number of barriers, access to the arena's center, and illumination on six movement properties. We demonstrate great differences among arenas. For example, the beetles moved over longer distances in clear arenas than in obstructed ones. Movement along the arena's perimeter was greater in smaller arenas than in larger ones. Movement was more directional in round arenas than in rectangular ones. In general, the beetles stopped moving closer to the perimeter and closer to corners (in the square and rectangular arenas) than expected by chance. In some cases, the arena properties interacted with the beetle sex to affect several movement properties. All these suggest that arena properties might also interact with experimental manipulations to affect the outcome of studies and lead to results specific to the arena used. In other words, instead of examining animal movement, we in fact examine the animal interaction with the arena structure. Caution is therefore advised in interpreting the results of studies on movement in arenas under laboratory conditions and we recommend paying attention also to barriers or obstacles in field experiments. For instance, movement along the arena's perimeter is often interpreted as centrophobism or thigmotaxis but the results here show that such movement is arena dependent.


Introduction
Movement is an integral component of many animal behaviors, like foraging, predation avoidance, dispersal, and mate finding.Even plants move, either passively by wind or water, for example, or actively, following the sun's position (Forterre, 2013;Kutschera & Briggs, 2016).Movement, defined as a temporal change in position, involves Correspondence: Inon Scharf, George S. Wise Faculty of Life Sciences, School of Zoology, Tel Aviv University, 69978 Tel Aviv, Israel.Tel: +972 3 6408006.Email: scharfi@tauex.tau.ac.il a variety of questions, such as why, how, when, and where to move (Nathan et al., 2008).Like many other behaviors, movement is dictated by intrinsic and extrinsic factors.Hunger level is an important intrinsic factor, and movement usually increases with hunger but then decreases following a hump-shaped pattern (reviewed in Scharf, 2016).Predation risk is a common example of an extrinsic factor, which typically decreases the prey foragingrelated movement but triggers other movements, those related to escaping predators (Sih, 1984;Gottlieb et al., 2017;Richardson et al., 2018).Movement can be directly measured, when individual animals are followed, either by sight or other means, such as radiotelemetry, and their positions are documented, or indirectly, for example, by clearing an area and observing animal footprints after a fixed time (Ward et al., 2013;Subach, 2020).
There are multiple ways to characterize the movement (Turchin, 1998, ch. 5).The most obvious measure is the total distance moved, which is often used as a proxy for activity level.Other common measures are speed (distance/time), acceleration (speed changes), directionality (turn rate), or the autocorrelation in time of the listed measures.Characterizing movement is important for many purposes.For example, the switch between a directional movement and a more tortuous one is typical for area-restricted search, which is an efficient foraging method when prey is clumped (Benhamou, 1992;Dorfman et al., 2022).The energy spent during movement is relevant when referring to the benefits and costs of foraging (Pyke et al., 1977;Louzano et al., 2014).It increases with movement distances but also increases per time unit together with movement tortuosity and speed (Wilson et al., 2013(Wilson et al., , 2015)).
Many experiments in the laboratory or under semifield conditions, like enclosures, record animal movement for different reasons.For example, in vertebrates, movement is often documented to examine the proportion of movement next to the test arena's walls, interpreted as anxietylike behavior (Simon et al., 1994;Harris et al., 2009;Johnson & Hamilton, 2017).Notable various behaviors differ when measured under natural conditions and in the laboratory.For example, cannibalism, same-sex sexual behavior, and the response to predation risk are sometimes stronger under artificial conditions than under natural ones (Kuba & Koyama, 1985;Abramsky et al., 1996;Wilder & Rypstra, 2012).Only a few studies compared movement under artificial and natural conditions.An exceptional study determined that squirrel monkeys reduce their gait flexibility in the laboratory compared to nature (Shapiro et al., 2010).The fact that some behaviors differ under natural and artificial conditions is not surprising.When the behavioral responses measured in nature and the laboratory are correlated, the experimental results in the laboratory are valid.However, it is intriguing to examine how the chosen conditions in the laboratory can influence behavioral responses.It is clear, for example, that the illumination level can affect activity in general and movement, in particular (Britz & Pienaar, 1992;Scharf et al., 2008;Russart & Nelsson, 2018).
Since the research on animal movement is often done in the laboratory, the strength of laboratory artifacts is important to study.For example, studies on various taxa, like rodents, ants, and flies have examined movement in enclosures and documented wall-following behavior.In some such studies, the enclosures were round (e.g., Gonzalez et al., 2017;Scharf et al., 2021) whereas in other studies they were rectangular (e.g., Dussutour et al., 2005;Avni & Eilam, 2008;Hänzi & Straka, 2018).Even studies related to wall-following behavior on a single species, Drosophila melanogaster, for instance, differed in the enclosure shape (round vs. rectangular;cf. Chen et al., 2014;Kottler et al., 2019;and Besson & Martin, 2005;Leberton & Martin, 2009).Even when the same shape is used, for example, a round arena, to test D. melanogaster, the enclosure often differed in size (e.g., r = 2, 3, 3.5, 4.4, or 9 cm; Götz & Biesinger, 1985;White et al., 2010;Ismail et al., 2015).Another good example is that of shoaling behavior in zebrafish, which has been studied in arenas differing in size and shape (reviewed in Buske, 2015).This inconsistency might be of little importance if the enclosure shape and area would not affect important movement properties.If, however, the enclosure shape/area dramatically affects movement properties, it might put into question differences determined either as significant or not in laboratory studies.For example, an enclosure that triggers much movement might exaggerate minor differences among treatments and make them significant, and vice versa: an enclosure that triggers little movement might miss important differences among treatments due to a low effect size.Another example of differences in the arena's structure, which can affect movement, takes place when small barriers are placed in the arena but in a different spatial distribution pattern, either regular or random (Grez & Villagrán, 2000;Yaski et al., 2011a,b).
Our main goal here is to conduct a comprehensive examination of whether and to which extent the enclosure characteristics affect important movement properties.Although some reports on such effects exist, especially in mice, rats, and zebrafish, there is currently no rigorous investigation of this question and very little information on invertebrates.Specifically, we examined in the laboratory how the test arena size and shape affect movement distance, the proportion of time active, wallfollowing behavior, turn angles or movement directionality, and autocorrelation in time of both movement steps and turn angles.We used the red flour beetle, Tribolium castaneum, as the study animal.This beetle is an important pest of stored products and is an excellent disperser (Ridley et al., 2011;Atta et al., 2020).There is much research on its activity, dispersal, habitat selection, and patch colonization success (Campbell & Hagstrum, 2002;Halliday & Bloiun-Demers, 2014;Arnold et al., 2017a;Matsumura et al., 2022).Movement is an important component of all of the above and it is therefore important to study it.The red flour beetle has served as a model organism in various biological research fields, both pure and applied science (Schröder et al., 2008;Brown et al., 2009;Campbell et al., 2022).On top of that, several previous studies used arenas of different structures to measure its movement properties in the laboratory (e.g., Romero et al., 2010;Wexler et al., 2016;Matsumura & Miyatake, 2018;Scharf et al., 2019;Sakka et al., 2020).Owing to the evidence for the effect of illumination on the studied species (Arbogast & Flaherty, 1973;Song et al., 2016), we examined also how illumination should affect movement properties.

General methods
Red flour beetles, T. castaneum (Herbst, 1797), were collected in early 2020 in mill storage in northern Israel and brought to the Ministry of Agriculture.In March 2020, they were brought to Tel Aviv University and kept in a climate cabinet at 30 °C (the optimal temperature: Halliday & Blouin-Demers, 2014), 12 : 12 L : D, for at least 25 generations.To create a new generation, we selected 500 random beetles and placed them in five round boxes (diameter of 11 cm) filled with 120 g of a wheat flour-yeast mixture (ratio of 10 : 1).The beetles from the different boxes were mixed every three generations.The experiments were conducted in October 2022−January 2023.We separated pupae according to sex, placed them in a similar flour-yeast mixture, and allowed them to emerge.The experiment commenced two weeks following the separation of pupae.Ten individuals per sex took part in each experiment.Each experiment comprised three treatments, that is, different arenas or illumination levels.Each beetle was tested under all treatments, in random order.The beetles were tested in arenas with white paper glued on their surface to improve the contrast while filming and allow the beetles to move more easily.Each beetle was placed in the arena center, covered with a tube lid (3 cm in diameter) for 30 s, for acclimation.We then removed the lid and the test commenced.We filmed the beetle for 5 min (using Logitech Brio 4K Ultra HD Webcam), 30 frames/s.After each test, the beetle was moved to a small plate with a bit of flour on which it could feed.Each dish was aired between tests for 25−35 min before being reused.All experiments were conducted in the afternoon (12:00−16:00).

Experiments
Each experiment comprised three treatments (see Fig. 1 for a scheme).
Experiment 1: arena size We used round Petri dishes of three diameters: 5.8, 9.2, and 14.3 cm (26.4,66.5, and 160.6 cm 2 , respectively).The arenas differ in size but also in the angle of the circular arc (more obtuse in larger arenas).
Experiment 2: arena shape We used a Petri dish of 9.2 cm in diameter, a square (8.3 cm × 8.3 cm), and a rectangle (12 cm × 5.8 cm).All three are almost identical in their surface area (66.5−69.6 cm 2 ), so they differ mostly in shape (corners vs. no corners and edge length) but also in their perimeter.
Experiment 3: arena's center accessibility We used Petri dishes of 14.3 cm in diameter, either empty, with a 5.8 cm plate glued in the center creating a broadaccessible ring, or a 9.2 plate glued in the center creating a narrow-accessible ring.Whereas the dishes in Experiment 1 differed both in size and the angle of the circular arc, here the perimeter was identical in size and shape, but the arena's area differed (160.6, 134.2, and 94.1 cm).If the beetles move mostly along the perimeter, preventing access to the arena's center should have only a minor effect on their movement.In other words, an inaccessible center is a method to reduce the arena size without interfering with the arena's perimeter.
Experiment 4: barrier number We used Petri dishes of 14.3 cm in diameter, either empty or with two or six thin walls of one cm in length each.We hereafter refer to these walls as "barriers" because they intervene with the beetles' movement.Indeed, if the beetles move mostly along the perimeter, barriers on the perimeter should interfere strongly with the beetles' movement.
Experiment 5: illumination level We used a Petri dish of 9.2 in diameter and changed the illumination level in the laboratory during the experiment: 337 Lux, 121 Lux, or 14.5 Lux.Illumination may increase activity, as the beetle may be attracted to light (Soderstorm, 1968).

Movement analysis
The beetle positions (x, y coordinates) in each video frame were obtained using the free software ToxTrac (Rodriguez et al., 2018).Movement properties were identically analyzed in all experiments.We documented six movement properties.(1) Movement distance: We summed the movement distance between each pair of coordinates.The result was calculated in pixels and divided by the known arena size to obtain the distance in cm.(2) Movement along the arena's perimeter: We calculated the proportion of beetle positions, which are 0.5 cm or less from the arena's perimeter.We did so every 10th frame (900 values).We decided to use a threshold of 0.5 cm based on previous studies on the same species using a similar threshold of 0.45 cm (Wexler et al., 2016;Wexler & Scharf, 2017).We decided against using a proportion of the arena (e.g., the peripheral 5%) because we used different arena sizes and shapes and wished to have a consistent perimeter width across arenas and experiments.(3) Number of stopping events: Number of events during which the beetle moved less than 5 mm in 3 s (reported by ToxTrac).( 4) Turn angles: To calculate turn angles, we referred only to every 10th frame.We believe this is a suitable compromise between over-and undersampling (Turchin, 1998, ch. 5).We calculated the median value of all angles, each calculated as the angle among three successive coordinates, creating a triangle.The angles were referred to in absolute values to equally treat right and left turns (as we were not interested in laterality).The angle of keeping the current movement direction was set to 0°. (5) Autocorrelation of movement steps: We calculated the Pearson's correlation between each pair of successive movement steps at time t and t + 1, using the collapsed data of 3 frames/s.(6) Autocorrelation of turn angles: We calculated the Pearson's correlation between each pair of successive turn angles at time t and t + 1.The first indicates how consistent the beetles are in movement distances between successive steps (i.e., whether they keep moving at the same speed or often change it).The second indicates whether the beetles keep moving in the same direction between successive steps (a higher autocorrelation) or frequently change directions (a lower autocorrelation).

Statistical analysis
All experiments were identically analyzed.We analyzed each of the six above-mentioned response variables separately and used a repeated-measures ANOVA with the treatment as the within-subjects variable, and sex as the between-subjects variable.When neither sex nor its interaction with treatment was significant, they were removed, and the test was redone.Each response variable was checked before the analysis to see whether it deviated from a normal distribution.If so, we used a square-root transformation.The proportion of movement along the arena's perimeter was arcsine-transformed, as common for proportions (Gotelli & Ellison, 2004, ch. 8).To examine correlations among the six movement properties, we conducted a principal component analysis (PCA) for each experiment separately (after a z-score transformation).We refer to loadings higher than 0.3 or lower than −0.3 as significantly correlated (Tabachnick & Fidell, 2007).The statistical analysis was done with SY-STAT v. 13 (Systat Software, Inc., San Jose, CA, USA) and the PCA with Matlab (MathWorks Inc., Natick, MA, USA).

Experiment 1: arena size
Beetles moved over longer distances in the large arena than in the medium one, and more in the medium arena than in the small one (Table 1A; Fig. 2A).Beetles moved more frequently along the arena's perimeter in the smaller arena than in the medium arena, and more in the medium arena than in the larger one (Fig. 2B).The number of stopping events did not differ among treatments, but beetles stopped more frequently along the perimeter than expected by chance (Supplementary material, part If neither sex nor its interaction with treatment was significant, it was removed and the analysis redone (this changed the error df of the treatment from 36 to 38).Significant results appear in bold and marginally significant results in italics.
2).Movement directionality was greater (or turn angles lower) in the large arena than in the small one (Fig. 2C).
Regarding the autocorrelation of movement steps, beetles in the large arenas were more consistent in their movement steps (constant speed) than those in the small arena (Fig. 2D).Treatment, however, did not affect the autocorrelation of turn angles.Neither sex nor its interaction with treatment affected any of the response variables.

Experiment 2: arena shape
There was a marginally nonsignificant difference in movement distances among shapes (Table 1B) with the least movement in a rectangular one.Beetles in the squared arena tended to move more along the arena's perimeter than in the rectangular one (Fig. 3A).The number of stopping events did not differ among treatments.However, beetles stopped closer to the corners than expected by chance (Supplementary material, part 3).Movement directionality was higher in the round arena than in the rectangular arena (Fig. 3B).There was no effect of arena shape on the autocorrelation of movement steps, but the autocorrelation of turn angles was much higher in the round arena than the two other ones (Fig. 3C).Neither sex nor its interaction with treatment affected any of the response variables.The beetles moved over greater distances, (B) moved less along the arena's perimeter, (C) their movement was more directional (lower turn angles), and (D) their movement steps were more consistent (the autocorrelation was higher) in the large arena than in the small one.The boxplots show the mean (×), the first and third quartiles (a rectangle), the median (a horizontal line within the rectangle), and the whole range without outliers.Outliers are not shown.

Experiment 3: arena's center accessibility
Treatment had no effect on movement distances, preference for the arena's perimeter, the number of stopping events, and the autocorrelation of movement steps (Table 1C).Females tended to stop more frequently (a marginally nonsignificant difference).Directionality level differed among treatments (Fig. 4A): movement was the most directional in the control, followed by the movement in the broad ring treatment, and the movement in the narrow ring treatment was the least directional.The autocorrelation of directionality was the highest in the control and lower in both ring treatments.However, there was also a significant treatment × sex interaction (Fig. 4B): Whereas in the control and broad ring treatments females and males behaved similarly, males in the narrow ring treatment were more consistent in their movement directions than females.

Experiment 4: barrier number
Both treatment and sex affected movement distances (Table 1D).Movement distances were greater in the control arena than in the two arenas with barriers and males moved more than females (Fig. 5A).Beetles tested in the control arena also moved more on the arena's perimeter than in the six-barrier arena while the two-barrier arena was in-between (Fig. 5B) with no effect of sex.The number of stopping events was lower in the control than in the six-barrier arena with the two-barrier arena in-between and females ceased movement more than males.Movement directionality was higher in the control than in the six-barrier treatment and did not differ between the sexes (Fig. 5C).The autocorrelation of movement steps was greater for females than males but was not affected by the arena.Finally, the autocorrelation of turn angles was higher in the control than in both other arena treatments with no effect of sex (Fig. 5D).Experiment 5: illumination level None of the explanatory variables (treatment, sex, or their interaction) affected movement distances, the proportion of movement along the arena's perimeter, the number of stopping events, turn angles, or autocorrelation of turn angles (Table 1E).However, treatment and sex interacted to affect the autocorrelation of movement steps: it was greater in males under the darkest conditions, whereas the opposite held true under the most lit conditions (Fig. 6).

Correlations among movement properties
Overall, PC1 explained between 39.8% of the variance in Experiment 3 to 46.6% in Experiment 4. The emerging pattern was similar in all experiments (Table 2A): Movement distance loading positively and both the number of stopping events and turn angles loading negatively.In other words, the longer beetles moved the less often they stopped, and their movement was more directional.In three of the five experiments, the autocorrelation of  movement steps loaded positively on PC1, indicating that movement over longer distances usually goes together with more consistent movement steps.PC2 explained between 19.4% of the variance in Experiment 4 and 27.8% in Experiment 5.Here too, the emerging pattern was similar in most experiments (Table 2B): The autocorrelation of turn angles loaded positively in all experiments and movement along the arena's perimeter as well as the autocorrelation in movement steps loaded positively in all but one experiment.We interpret it to mean that movement along the perimeter increases consistency in both movement steps and turn angles.

Discussion
Movement properties of flour beetles in arenas of distinct sizes, shapes, and configurations differed greatly.It is not only that beetles moved over greater distances in some arenas than others, but also other movement properties differed, such as the directionality level or the extent of movement along the arena's perimeter.For example, enlarging the arena led to longer movements, increased movement directionality, and decreased the proportion of movement along the arena's perimeter.Changing the arena from round to rectangular decreased movement directionality and placing small barriers along the perimeter changed almost all movement properties documented.We will discuss below the specific changes in behavior but would like to emphasize first the meaning of testing animal movement in the laboratory: Care should be taken to verify that the chosen arena fits the study animal and that the tested treatments do not interact with the arena chosen to affect various movement properties.That said, the link among different movement properties was similar across experiments.Movement distances were negatively correlated with the number of stopping events and with turn angles, and movement along the arena's perimeter was positively correlated with the consistency in movement steps and turn angles.This suggests that although different arenas are used, movement can be characterized using similar properties (as shown also in Arnold et al., 2017a).

Movement distances
Beetles moved over greater distances in larger round arenas in Experiment 1.However, a greater movement was not observed in the larger arenas in Experiment 3 (accessible or inaccessible arena center).This suggests that movement distance does not depend so greatly on the arena's size but rather on the angle of the arena's perimeter, as it is different in Experiment 1 among arenas but is not different in Experiment 3. The comparison between the two experiments suggests that movement distance is greater when the angle of the circular arc is more obtuse (compare arenas in Experiment 1).That said, a straight perimeter, as the square in Experiment 2, did not lead to greater movement, indicating that the angle of the arena's perimeter does not fully explain movement distances.Several previous studies suggested that the arena size might affect movement distances.For example, Colwill and Creton (2011) suggested that the small arenas used in their experiment with zebrafish might have limited exploration and have limited their ability to detect significant differences among treatments.In contrast, Eilam et al. (2003) found no effect of the size of square arenas on the movement distances of voles.Flour beetles are excellent dispersers and the distance moved is tightly linked to dispersal success.Arnold et al. (2017b) examined dispersal apparatuses of different insertion angles forcing the beetles to climb.They showed great differences in dispersal success based on such angles.Similar to our study regarding the movement in arenas, caution is required in how dispersal success is interpreted when the beetles are examined in different apparatuses.Another fine example of a behavioral change is the change in the functional response of ladybird beetles, used in biological control, with arena size (Uiterwaal & DeLong, 2018).

Movement along the arena's perimeter
Movement along the arena's perimeter was generally high (over 80%).Moving on the perimeter may stem from different reasons.For example, some animals do so if they are stressed or anxious, and others use the perimeter as a prominent landmark, especially in the absence of other landmarks (Avni & Eilam, 2008;Harris et al., 2009).The frequent movement along the perimeter suggests that manipulating the structural changes in the arena's perimeter should affect movement more than changes in its center.Indeed, changing the arena's center accessibility led to fewer differences in movement than those induced by changing the perimeter shape or adding barriers on the perimeter.Movement along the perimeter was somewhat less frequent in the rectangular arena than in the square one (Experiment 2).Stewart et al. (2010) showed a difference between round and rectangular arenas in the proportion of movement in specific areas defined as base," mostly next to the arena's perimeter.Thus, there too, the arena shape affects spatial behavior.Movement along the perimeter is not just a function of the existence of corners, as the deviating shape was a rectangle whereas round and square arenas resembled more one another.Movement along the perimeter decreased with the increase in the number of barriers in the arena (Experiment 4).This result suggests that wall-following by flour beetles is passive rather than active, similar to a cockroach or a frog species examined (Creed & Miller, 1990;Hänzi & Straka, 2018) and unlike a cavefish (Patton et al., 2010).If it were active, then the beetles would move around the barrier and resume movement along the arena's perimeter (Creed & Miller, 1990).In contrast to our finding, that beetles moved less along the perimeter in large arenas than in smaller ones, Eilam et al. (2003) found that voles traveled more often along the walls in large arenas vs. small ones.This might suggest that wall-following is triggered by different mechanisms in the two studies.Yaski et al. (2011a) found that rats move longer along the perimeter in round arenas than in square ones.Here, the main difference was between a square arena and a rectangular one, supporting once more the suggestion that a different mechanism is involved.

Movement directionality and the number of stopping events
Movement directionality was higher (or turn angles were lower) in larger arenas than smaller ones and in the round arena than in the square and rectangular ones.
It also decreased with the increasing number of barriers (Experiment 4).The latter result is not surprising, as the beetles reaching a barrier had to bypass it and were forced to turn.Turns are more costly than moving in straight lines and are more costly the faster animals move (Wilson et al., 2013;McNarry et al., 2017).Our analysis also indicates that sharp turns are correlated with slower movement or shorter steps (Supplementary material, part 1).Thus, beetles turned, slowed down, and might have stopped more frequently as a result, expressed here as a greater number of stopping events.The movement was more directional in larger round arenas because as beetles followed the arena's perimeter, their turn angles were more acute than in smaller round arenas.Thus, we would have expected to observe the most directional movement in square or rectangular arenas, which did not hold true.In contrast, the movement was less directional in the latter two than in the round arena (Experiment 2).The reason may be the higher beetle tendency to follow the perimeter when it is concave than straight (cf. the round and rectangular arenas), similar to rats (Yaski et al., 2011a,b).Limiting access to the center decreased movement directionality, probably because the beetles encountered the inaccessible center and bounced back, resulting in more frequent angle changes.Regarding the stopping events, we received a mixed pattern.On the one hand, barriers (Experiment 4) increased the number of such events, suggesting an exogenous cause for stopping.On the other hand, although the beetles stopped more frequently close to corners, they did not stop more in square or rectangular arenas than in round ones, suggesting that stopping is also an endogenous mechanism.Thus, the arena structure may perhaps change the explanation of stopping from mostly exogenous to mostly endogenous.This suggestion remains to be tested.

Autocorrelation of movement steps and turn angles
Successive movement steps were more similar in length in the large arena than the small one (Experiment 1), indicating that in the former one, the beetles use more constant speed.Repeatedly changing speed is costlier than moving at a constant speed (Seethapathi & Srinivasan, 2015), suggesting that movement in a small arena is costlier per unit of distance.The beetles kept moving in similar directions in the round arena more than in the square or rectangular ones (Experiment 2), explaining perhaps also why movement in the latter two was more tortuous.Once more, a concave perimeter perhaps leads to more movement along the perimeter and hence also less frequent turns.Blocking the access to the arena's center (Experiment 3) disrupts the consistency of movement direction probably owing to frequent encounters with the blocked center, which bounced the beetles back to the perimeter.Similarly, barriers on the perimeter forced the beetles to turn when encountering them ending in the same lower consistency in movement directionality.The illumination level had a moderate effect on the autocorrelation in movement distances, interacting with sex.As light is known to affect this species' behavior, we suggest that the experiment was either too short to demonstrate effects by illumination or that the differences among treatments were not sufficiently large.This remains to be tested.

Conclusion
We demonstrate here that the physical characteristics of the test arena often alter several movement properties of the studied beetle.On top of that, we detected several two-way interactions of the arena's traits with sex affecting movement properties.This fact suggests that such physical properties might interact with other treatments or experimental manipulations of interest, and potentially bias the results of similar laboratory experiments.For example, arenas that trigger low movement might impair the detection of significant differences among treatments, or experiments in arenas that enhance movement along the perimeter might be interpreted as strong thigmotaxis or centrophobism.We recommend examining animals in more than a single enclosure or conducting preliminary examinations to determine which enclosure would result in the least possible bias.Future studies may examine how the arena interacts with experimental manipulations, such as the body condition, life stage, or density.For example, the studied beetles move over shorter distances with age, starvation, when their legs are shorter, and following repeated cold shocks (Wexler et al., 2016;Arnold et al., 2017a;Scharf et al., 2019).It is yet to be uncovered whether such effects are robust or depend on the specific test arena characteristics.Our conclusions are not limited to the laboratory and are probably relevant to field experiments too.The physical characteristics of the habitat can interact with experimental manipulations to affect the spatial behavior of studied species and such possible effects should be considered.

Fig. 1
Fig. 1 A scheme of the experimental design of the five experiments.(A) Arena size: Three round arenas of different sizes (diameter of 5.8, 9.2, and 14.3 cm).(B) Arena shape: Three arenas of different shapes [round (d = 9.2 cm), square (8.3 cm × 8.3 cm), and rectangle (12 cm × 5.8 cm)].(C) Arena's center accessibility: Reducing the arena's size (d = 14.3 cm) without changing the angle of the arena's perimeter.The black area was inaccessible to the beetles (either d = 5.8 or d = 9.2 cm).(D) Number of barriers: Increasing the barrier number (none, two, or six barriers) on the perimeter of the arena (d = 14.3 cm).(E) Illumination level: either 337 Lux, 121 Lux, or 14.5 Lux.Arenas of 9.2 cm in diameter.Each beetle was tested in all treatments of a single experiment in random order.

Fig. 2
Fig.2Differences in movement properties with arena size.(A) The beetles moved over greater distances, (B) moved less along the arena's perimeter, (C) their movement was more directional (lower turn angles), and (D) their movement steps were more consistent (the autocorrelation was higher) in the large arena than in the small one.The boxplots show the mean (×), the first and third quartiles (a rectangle), the median (a horizontal line within the rectangle), and the whole range without outliers.Outliers are not shown.

Fig. 3
Fig. 3 Differences in movement properties with arena shape.(A) Movement along the arena's perimeter was lower in the rectangular arena than in the square one.(B) Movement was less directional (higher turn angles) in the rectangular arena than in the round one.(C) The autocorrelation of turn angles between successive steps was higher in the round arena than in the other two.

Fig. 4
Fig.4Differences in movement properties when blocking the accessibility to the arena's center.Narrow ring and broad ring indicate that larger and smaller parts of the arena are blocked, respectively.(A) Movement was the most tortuous (higher turn angles) in the narrow ring and the most directional (lower turn angles) in the control.(B) Males were more consistent in their movement directionality than females in the narrow ring treatment whereas there was no difference between the sexes in the two other treatments.Generally, the turn angles were more consistent in the control than in the two other treatments.

Fig. 5
Fig. 5 Differences in movement properties with the number of barriers in the arena.(A) The distance moved and (B) the movement along the arena's perimeter were greater in the absence of barriers.(C) Turn angles were lower in the absence of barriers (more directional movement).(D) The autocorrelation of turn angles between successive steps was also greater in the absence of barriers.

Fig. 6
Fig.6Differences in movement properties with the interaction of illumination level and sex.Females were more consistent in their movement steps under the two higher illumination levels, while males (right columns, orange) were under the lowest illumination level.

Table 1
All statistical results in Experiments 1−5.

Table 2
PCA results on the six movement properties in each of the five experiments.Loadings larger than an absolute value of 0.3 are marked with gray.