Disruption of social play influences development of caution in juvenile ground squirrels

Disruption of play behavior influences development of caution in juvenile ground squirrels Methods General Methods A population of Belding’s ground squirrels (Urocitellus beldingi) was studied from June-August in 2021 and 2022 at Tioga Pass (latitude 37.9, longitude -119.2, elevation 2950 meters) in Mono County, California, USA. We captured squirrels with live-traps (Tomahawk Live-Trap, Hazelhurst, WI) baited with peanut butter. We checked traps every thirty minutes or less during trapping sessions, and released squirrels at their sites of capture after handling. We fitted squirrels with numbered metal ear tags for long-term identification (National Band and Tag, Newport, KY). We marked squirrels marked with symbols using hair dye (Revlon, New York, NY) to aid in identification of individuals during observations and behavioral tests. To further aid in identification of individuals we painted ear tags of juveniles different colors with nail polish (Nunes et al. 2015). At juveniles’ first capture, we weighed squirrels were weighed to the nearest gram with spring balance scales (Avinet Research Suppliers, Portland, ME). We observed the maternal territories of reproductive females daily to determine the dates that their litters first emerged from the natal burrow. We trapped, ear-tagged, and weighed juveniles within two days of first being observed above ground. A total of 71 juveniles from 12 litters were trapped in 2021, and 55 juveniles from 9 litters were trapped in 2022. Trapping and other methods in the study followed guidelines published by the American Society of Mammalogists (Sikes et al. 2016). Observation of Behavior We regularly observed the behavior of juvenile U. beldingi throughout the day between 0730-1400 hours from elevated posts such as boulders or hillsides. Observation sessions lasted from 30-120 minutes and were terminated when juveniles became scattered throughout the natal area and were no longer interacting with each other. Most observation sessions occurred in the morning between 0730 and 0930 after juveniles first emerged from the natal burrow for the day and all littermates were present and active. During observations, we recorded all occurrences of social play behavior for individual U. beldingi along with the duration of the interactions. Specific play behaviors are described in Nunes et al. (1999). Rates of social play were calculated for individual U. beldingi as the number of interactions observed per hour of observation. Juveniles included in the study were observed for an average of 338.2 + 15.2 (SE) minutes per individual on an average of 4.9 + 0.2 (SE) different days from 2-11 days after their first appearance above ground. Disruption of Behavior At their first emergence from the natal burrow, 21 litters, each with 5-7 juveniles, were randomly assigned to one of three groups: control/undisrupted (7 litters, 40 juveniles), non-play-disrupted (7 litters, 44 juveniles), or play-disrupted (7 litters, 42 juveniles). We note that a total of 8 juveniles disappeared during their first two weeks above ground (3 undisrupted, 2 non-play-disrupted, 3 play-disrupted,) and were omitted from the study. Follow-up behavioral tests (see below) could not be completed for three juveniles in the play-disrupted group, and these juveniles were also omitted from the study. The final sample consisted of 115 squirrels, including 37 undisrupted juveniles (20 female, 17 male), 42 non-play-disrupted juveniles (24 female, 18 male), and 36 play-disrupted juveniles (20 female, 16 male). Juveniles in the undisrupted group were observed during observation sessions as described above without interference. During observations of juveniles in the non-play-disrupted group, every five minutes an observer walked at a pace of one meter per second through areas in which juveniles were present. If any juveniles were engaged in play interactions at the time of a walk-through, the observer waited until the interactions were completed before walking though the area. During observations of play-disrupted juveniles, an observer disrupted all play interactions by walking at a pace of one meter per second toward the juveniles engaged in play until the play-bout was halted. Juveniles in the non-play-disrupted and play-disrupted groups were also observed without disruption for at least one observation session to determine baseline rates of play and baseline duration of play bouts. Behavioral Tests To evaluate caution in response to an intruder in the natal area, behavioral tests were conducted toward the beginning of the play period within 1-3 days after individuals first appeared above ground, and again toward the end of the play period within 12-14 days after first emergence from the natal burrow. Behavioral tests were a modified version of flight-initiation distance tests (Runyan and Blumstein 2004; Nunes 2023), which measure the distance at which an individual first flees from an approaching human (Ydenberg and Dill 1986; Blumstein 2003). Flight is an anti-predatory behavior, and flight-initiation distance is commonly used to measure responses along the caution-boldness continuum (Cooper 2009; Petelle et al. 2013). During tests, a human intruder identified a test subject and took a position approximately 20 meters from the subject. At the initiation of tests, the juveniles being tested (1) had been facing away from the intruder and for at least two minutes, (2) was not within two meters of any other squirrels, and (3) was within 1.5 meters of a burrow entrance that could be used for escape. The intruder walked toward the subject at a pace of one meter per second and dropped a marker flag (1) when the squirrel first noticed the intruder, as indicated by the squirrel turning toward the intruder, lifting its head or body, or standing on its hind legs, (2) when the squirrel fled from its original location, and (3) at the squirrel’s original location. Measuring tapes were used to determine distances from their original location at which squirrels noticed and fled from intruders. Changes in these distances across the play period were expressed as proportions by dividing distances at the end of the play period by those at the beginning. We conducted tests after juveniles had emerged from the natal burrow in the morning, had become scattered throughout the natal area, and were not interacting with other squirrels. In rare occurrences during which another squirrel responded to the intruder before the focal squirrel did, the test trial was terminated, and a new trial was conducted for the focal squirrel on the following day (Shehan et al. 2023). References Blumstein, D. T. 2003. Flight-initiation distance in birds is dependent on intruder starting distance. J. Wildl. Manage. 67, 852-857. https://doi.org/10.2307/3802692. Cooper W.E. Jr., 2009. Variation in escape behavior among individuals of the striped plateau lizard Sceloporous virgatus may reflect differences in boldness. J. Herpetol. 43, 495-502. https://doi.org/10.1670/08-197R1.1. Nunes, S., 2023. Animal-friendly behavioral testing in field studies: examples from ground squirrels. Front. Behav. Neurosci. 17, 1239774. https://doi.org/10.3389/fnbeh.2023.1239774. 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