Bears habituate to the repeated exposure of a novel stimulus, unmanned aircraft systems

Drones are powerful new tools used in several biological sciences. Previous work indicated that animals behave fearfully or show a stress response near drone flights. Using heart monitors to gauge stress, we found that bears habituated to drones after ~20 flights over a 3–4-week period and remained habituated.


Introduction
The human footprint continues to expand, reducing available habitat and increasing the frequency of interactions between wildlife and human activities (Venter et al., 2016). 1 responses of wildlife that may not be apparent from behavioral responses alone .
The popularity of unmanned aircraft systems (UAS; i.e. 'drones') among recreationalists, researchers and conservationists has increased tremendously in recent years (Anderson and Gaston, 2013) and represents a new potential stress to wildlife Mulero-Pázmány et al., 2017). As hurdles to use of UAS are eased Werden et al., 2015), UAS technology is seeing more use in population surveys (Seymour et al., 2017;Witczuk et al., 2017;Hodgson et al., 2018), collection of biological samples (Wolinsky, 2017;Domínguez-Sánchez et al., 2018) and fine-scale habitat data (Olsoy Peter et al., 2018), observations of behaviors (Schofield et al., 2017;Barnas et al., 2018b) and curbing poaching (Mulero-Pázmány et al., 2015;O'Donoghue and Rutz, 2016). In the near future, UAS will be able to track animals using thermal signatures or VHF tags (Bayram et al., 2016(Bayram et al., , 2017Cliff et al., 2015Cliff et al., , 2018 and UAS swarms will be used to automatically identify multiple individuals or search larger areas for the presence of wildlife efficiently (Allan et al., 2018).
However, UAS may disturb animals more than other aerial survey methods due to the very nature of what makes these devices useful: the ability to fly and hover at low altitudes. Indeed, numerous studies have observed responses of wildlife to UAS (Pomeroy et al., 2015;Vas et al., 2015;Brisson-Curadeau et al., 2017;Mulero-Pázmány et al., 2017;Barnas et al., 2018a) and the noise may impact non-target species (Scobie and Hugenholtz, 2016). Work by our group  documented acute stress responses in wild American black bears (Ursus americanus) to lowaltitude UAS flights. Bears in the study often did not display behavioral signs of fear (e.g. fleeing) but in extreme cases, their heart rates nearly quadrupled (162 beats per minute) compared to pre-flight baseline data (41 beats per minute). Here, we conducted experiments to address three follow-up questions that arose from our original study: (1) do bears habituate to the presence of UAS, (2) if so, over what timescale does tolerance develop and (3) does tolerance persist in the absence of exposure to the stimulus? Chronic impacts of the use of UAS are of biological and ethical concern, especially if UAS are used in wildlife surveys designed to minimally disturb animals.

Field methods
We flew an Iris+ model quadcopter UAS (3D Robotics, Berkeley, CA, USA) over five adult captive American black bears (three females and two males) fenced together in a 372-m 2 facility maintained by the Wildlife Science Center (WSC) in east-central Minnesota. The WSC is an educational, non-profit organization that provides husbandry and regular veterinary care to bears used for educational and research purposes. The study subjects had varied backgrounds prior to being housed at the WSC; three came from other captive facilities, two from the wild. The WSC hosts tours, and staff often work around the enclosure, so the bears were accustomed to both human presence and mechanical noises, such as lawnmowers.
We measured responses of bears using an implanted cardiac biologger developed for human use (Medtronic plc, Reveal XT -Generation2 with BearWare, Minnesota, USA; see Laske et al. 2018 for device details). We are cognizant that both the implantation of these devices and the experimental UAS flights constituted a disturbance to these bears. Wildlife research often causes some amount of disturbance or discomfort to study subjects that needs to be weighed against scientific gains (Putman, 1995;McMahon et al., 2012). Our research team has successfully implanted and deployed hundreds of these cardiac biologgers in wild American black bears to gain insights about bear physiology, responses to environmental stressors, and advancements in human medicine . The miniaturization of the cardiac biologging devices (Wilson et al., 2015;Laske et al., 2018), along with their tested use in humans, combined with the extraordinary healing abilities of bears (Iaizzo et al., 2012), enabled the animals to quickly and fully recover from device implantation. We are consistently looking to improve our methods to reduce the potential harm to individuals. Moreover, we have tracked a number of individual wild bears with implanted cardiac biologgers over successive years and checked their health annually, finding that they showed no differences compared to non-implanted bears in terms of body condition and reproduction (Laske et al., 2017. Given the rapidly expanding use of UAS as a tool to study a variety of aspects of wildlife ecology, we believed that the use of the biologgers was warranted because they enabled us to investigate physiological responses that otherwise would not have been possible, and our findings can directly help to inform best practices that could reduce animal disturbance in the long run (McMahon et al., 2012).
In March 2016, bears were anesthetized as part of an animal handling course at the University of Minnesota with a mixture of Ketamine-Xylazine and a biologger was implanted subcutaneously in a peristernal location using aseptic techniques. The devices recorded each heartbeat and provided 2-min averages of heart rate (HR) in beats per minute (bpm). Data from the devices were downloaded telemetrically when the bears were anesthetized a year later for transport to a new WSC facility. Later, we matched the timing of UAS flights with date/time stamps on the HR data. flights, with 10-min pauses between flights (4 weeks each season × 2 days per week × 5 flights per day = 40 flights per season). The flight plan for all 80 flights included three distinct locations over the enclosure where the UAS hovered in place ('loitering') for 30 s. We designed the flight path to maximize the areal coverage of the enclosure. On each flight, the UAS completed this circuit three times at an altitude of 15 m before returning to the launch site, which was not visible to the bears. From the edge of the bear enclosure, we recorded the maximum sound pressure levels (dB SPL; re 20 μPa, Root Mean Square (RMS), A-weighted) during 4 flights using an Extech model 407 750 sound level meter. The average maximum value measured for all loitering and transition maneuvers was 60.3 dB (range 54.3-70.4 dB).
We operated our UAS at a low altitude, specifically aiming to elicit a cardiac response from which we could monitor a process of habituation (attenuated response) over time following repeated exposure. Our primary concern was not to determine the distance or flight approach that causes a physiological response. While we acknowledge that UAS operators rarely fly this close to target species, we chose to do so because we anticipated that the captive bears, living in an environment with regular human disturbances, might require a greater stimulus to achieve the same physiological reaction as wild bears in our previous study , which was the baseline we were attempting to emulate. Because the HR data were not retrieved until the study was over, we could not adapt the study design to responses of the bears and hence needed to ensure that the initial flights over the bears would elicit a response.
Furthermore, we note that in selecting flight altitudes, UAS operators must balance the tradeoff between soughtafter image resolution and avoiding disturbance to wildlife species, but this is complicated by the fact that disturbance distance is unknown for many species due to varying aural capacities and hearing thresholds (Scobie and Hugenholtz, 2016). Additionally, the noise of the UAS operations on any given flight can be impacted by ambient conditions and the approach path of the UAS (Vas et al., 2015;Mulero-Pázmány et al., 2017;Rümmler et al., 2018) and species may respond physiologically but with no apparent behavioral change .
Methods were approved by the University of Minnesota's Institutional Animal Care and Use Committees (Protocol # 1002A77516) and were conducted in accordance with all relevant guidelines and regulations. Animal husbandry practices at the WSC were approved by the Institutional Animal Care and Use Committees (UMN-005).

Statistical methods
We calculated the 95% confidence interval of HR for each bear during the 30-min period prior to the first flight of each flight day and used the upper values as the pre-disturbance baseline. We calculated differences between baselines and the maximum HRs while the UAS was in flight for each of the five flights per day (MaxHRDiff). We created two linear mixed models using R package nlme (Pinheiro et al., 2017;R Core Team, 2018) with MaxHRDiff as the response variable in each. In Model 1, we tested whether the cardiac responses to each initial flight on a given flight day changed through time both within and between seasons. We used the first flight of each day because bears showed the strongest cardiac response to that flight. We regressed the MaxHRDiff of each first flight with Julian date and the interaction (Julian date × season) while accounting for individual differences using a random intercept based on bear identity and a random slope for Julian date. Our second model tested whether HRs changed within flight days or between the two seasons. We regressed MaxHRDiff with repetition number and the interaction of repetition number × season. We included a random intercept for bear identity. Two of the study bears were in a side enclosure during our first day of flights, so we excluded this day of data on these two bears from the analysis. We considered the bears to be habituated if the mean MaxHRDiff of the five bears was ≤10 bpm for consecutive flight days and did not subsequently increase for more than a day.

Results
All bears showed at least one strong HR elevation in response to the presence of the UAS overhead (max. increase above baseline [MaxHRDiff] observed during any flight exposures: X = 52.7, range = 41−73 bpm). Responses to the initial flights each day diminished through the spring season ( Fig. 1; Julian: βˆ= −1.38, SE = 0.28, P value <0.001) and bears were considered habituated by the third week of flights (flight days 5 and 6; Fig. 1). Although it was not until the fourth week (flight days 7 and 8) when most individuals exhibited HR increases <10 bpm (Fig. 1). Anecdotal behavioral observations corroborate these findings (Supplementary Video S1). When flights resumed in the fall, HR responses to first flights of each day were similar to those at the end of the spring season ( Fig. 1;
The use of biologging technology has increased and enabled researchers to address increasingly more complex questions about the physiological responses of animals subjected to diverse anthropogenic stimuli (Wilmers et al., 2015;Wilson et al., 2015;Madliger et al., 2018). Our experimental approach of pairing cardiac biologger technology with captive individuals enabled us to address novel questions by collecting physiological data at very fine time-scales (2-min averages) and repeatedly exposing individuals to the UAS stimulus at a regular schedule, which would not have been possible to execute with bears in the wild. The integration of physiological data into management and conservation is promising but nascent (Wilson et al., 2015;McGowan et al., 2017); here, we aimed to integrate these unique sources of data for improving the best practices of UAS use in wildlife conservation and research.
Given that wildlife are already repeatedly exposed to UAS for both research and conservation purposes, it is useful to know that bears could become habituated to frequent flights within a period of just a few weeks. Further UAS technological advancements will soon enable autonomous obstacle avoidance under forest canopies, regular tracking of VHFtagged individuals (Bayram et al., 2017;Cliff et al., 2015Cliff et al., , 2018 and multiple UAS working simultaneously to search out individuals (Allan et al., 2018), all of which suggest increased disturbance to wildlife. Whereas our results indicated that the initial stress response attenuated, meaning the stimulus became less disturbing, this could entail other maladaptive consequences. For example, bears may key on road noise to alert them to the danger of crossing roads ; their waning response to other mechanical sounds may reduce their wariness and expose them to increased risks.
The rate of habituation is likely to be species dependent. Bears in general habituate to frequent contact with people, showing less fear after repeated exposure (Beckmann and Berger, 2003;Wheat and Wilmers, 2016); accordingly, bears may be predisposed to becoming tolerant of novel disturbances. Furthermore, our study subjects likely already had high tolerances for human activities, due to regular exposure to mechanical equipment, human interactions, and nearby vehicular traffic. Indeed, these captive bears initially responded less to UAS exposure than did wild bearsalthough the difference was not as large as might be expected given the background noises in their captive setting (max HR increase range among wild bears = 47-123 bpm ; max HR increase range among captive bears = 44-78 bpm). Also, the rate of habituation in our study was likely more rapid than in the wild, where interaction with UAS may be less frequent. However, we note that the frequency of flights used in our study may not be extreme in comparison to situations where UAS are used as antipoaching tools (Mulero-Pázmány et al., 2014;O'Donoghue and Rutz, 2016), or where individuals are regularly surveyed in confined areas (e.g. haulouts [Pomeroy et al., 2015], nests [Weissensteiner et al., 2015], colonies [Hodgson et al., 2018;Ratcliffe et al., 2015] and island species [Ballaria et al., 2016]). Additionally, as UAS flight times and communication among UAS improves with technological advances, UAS will commonly be used to repeatedly record behavioral observations (Allan et al., 2018).
While our approach utilizing biologgers enabled us to examine fine temporal-scale cardiac changes, we echo the cautions of Bejder et al. (2009) against extrapolating this single metric of habituation to potential physiological responses that were not measured. Given the unknown chronic effects of continual disturbance (Wright et al., 2007), we strongly recommend that UAS users follow proper ethical guidelines (Hodgson and Koh, 2016) when operating these aircraft near wildlife. We also call on researchers to increase efforts to mitigate behavioral and stress responses, for example, by using quieter, fixed-wing craft when possible and conducting data collection at a sensor's maximum useful distance.

Supplementary material
Supplementary material is available at Conservation Physiology online.