Electronic Supplementary Material (ESM) s6

Electronic Supplementary Material (ESM)

Title: Adaptation of the Aesop’s Fable paradigm for use with raccoons (Procyon lotor): considerations for future application in non-avian and non-primate species Journal: Animal Cognition Authors: Lauren Stanton*, Emily Davis, Shylo Johnson, Amy Gilbert, and Sarah Benson-Amram *Corresponding author info: University of Wyoming, Email Address: [email protected]

Additional Details - Materials and Methods

Raccoon Housing and Maintenance

Our study was approved by the USDA National Wildlife Research Center’s Institutional

Animal Care and Use Committee. All raccoons (N = 8) participated in other approved, non- invasive research protocols (QA-2111, QA-2290, QA-2492, and QA-2440) that were conducted concurrently with this study. Raccoon housing, maintenance, and procedures followed the standard operating procedure ACCO027.00 Raccoon Maintenance for OARF, and complied with

US law. Raccoons were housed individually in covered, outdoor enclosures measuring 3m x 3m x 2.5m with cement floors and mesh sidings. Each enclosure included a den box, metal table, litter pan filled with ground corncob, an elevated log, two metal bowls for food and water, and a burlap hammock. Occasionally raccoons were provided with additional enrichment items, including burlap sacks and plastic balls. The raccoons were not food deprived for this experiment, and had access to their food and water bowls during trials. None of the study subjects had previous experience with an Aesop’s Fable task, nor had they participated in any formal cognitive testing prior to this experiment.

Arm length measurements and water displacement

Before our study began, we obtained arm lengths of all study subjects by measuring along the inside of the forearms, from the tip of the longest digit to the intersection of the forearm and chest, which occurred when raccoons were anesthetized for procedures conducted under other

1 approved protocols. We filled the tube with 500mL of water, which was altered ±100mL depending on the arm length of the individual being tested. Therefore, the floating reward was always just out of reach for all participants, regardless of their arm length. We used one marshmallow cut into smaller pieces, instead of one whole marshmallow, as our floating reward to minimize the possibility of the stones pushing the reward to the bottom of the tube.

Experimental procedure

Participation in trials was voluntary, and all Phase I trials for each raccoon (N = 8) were conducted over three consecutive days between the hours of 18:00 and 06:00, when raccoons were most likely to be active. The raccoons were tested individually in their home enclosures and we set up a Sony Handycam (HDR-CX405) camera and a desk lamp outside of the enclosures to record all trials. The desk lamp only illuminated the lower front area of the enclosure, leaving the back and top of the enclosure dark. The desk lamp contained a 60 watt bulb and was not brighter than the natural sunlight raccoons were exposed to daily, nor the artificial lighting in the building that raccoons were occasionally exposed to. Trials began once the experimenter (L.S.) had placed all experimental items (i.e. Aesop’s Fable apparatus and stones/objects) in the enclosure, and ended after at least 20 minutes had passed. L.S. left the building after the trials began to encourage participation by the subjects. Trial length varied slightly because it was not always possible to enter the enclosure or remove the apparatus due to the behavior of the raccoons (e.g., raccoons would not move away from the enclosure door, held onto the apparatus, hid the stones).

Habituation and trials

Other Aesop’s Fable studies begin by training their subjects to use a platform apparatus, where the animals are trained to drop stones onto a collapsible platform to retrieve a food reward. Training is followed by > 10 habituation trials, where subjects are presented with the

2 apparatus and other testing materials with pieces of food on it (e.g., Jelbert et al. 2014; Miller et al. 2016). Unfortunately, our time working with the raccoons was limited, since two of the other concurrent protocols (QA-2290 and QA-2440) required euthanasia of the raccoons by a predetermined date. Thus, we were unable to implement separate training and habituation periods. We also wanted to provide raccoons with a chance to solve the task on their own, without training, since this is the first time this paradigm has been given to a non-human, mammalian species. Because we were exposing the raccoons to the apparatus and stones early on

(i.e., in the three initial trials), we decided the next trials would involve streamlining habituation and training procedures. We did this by placing small pieces of food (i.e., pieces of marshmallow, raisins, and/or omnivore chow) on the platform of the apparatus and on the stones balanced atop of the apparatus until the animals readily approached the apparatus and engaged with the stones. If raccoons still did not approach after three trials, we began placing additional food on the ground near the apparatus.

The stones used in Phase I were collected by L.S. and chosen based on the similar weight of each stone (mean = 85.63g, SE = 4.35) and the similar level of water raised when each stone was dropped into the tube (mean = 1.22cm, SE = 0.16). To retrieve the reward, raccoons needed to drop one or two stones into the tube. The ability of raccoons to reach into the tube varied among individuals, resulting in variation in the number of stones needed to obtain the reward. This was not something that we could account for, because the behavior of the raccoons when interacting with the apparatus varied considerably across individuals, and even across trials within an individual. For example, an individual that stood on their toes would be able to push its arm farther into the tube, increasing its reaching ability. Or, if an individual climbed on top of the apparatus and pushed its arm into the tube, it could reach farther by squeezing its shoulder into

3 the tube. If the raccoons failed to drop the stones into the tube during their three initial trials, they immediately progressed into learning trials for stone-dropping training.

If raccoons began independently dropping the stones into the apparatus during learning trials, and demonstrated this proficiency in at least two successive trials, their learning trials were completed. Because picking up the stones and placing the stones into the tube required substantial effort by the raccoons and could not be achieved accidentally, we felt that two consecutive trials demonstrated their general understanding of how to receive the reward. If raccoons did not meet this criterion, then they received a minimum of 12 learning trials. Raccoon

22 began dropping stones into the tube but struggled to obtain the reward and was therefore given six additional learning trials. Thus, the number of learning trials per subject (including

Raccoon 22) ranged from 10-18 trials (mean = 12.5).

To determine whether raccoons had successfully learned to solve the task, all subjects received a minimum of three final trials in which we presented raccoons with the apparatus and stones resting on the platform. Here, to retrieve the reward, raccoons had to pick up stones from the platform and drop them into the tube. If subjects successfully retrieved the reward in their three final trials, or showed continued interest in retrieving the reward (e.g., continued sniffing the apparatus, reaching arm into apparatus, handling stones), they received additional final trials.

If subjects successfully received the reward in two of six consecutive final trials, their training was completed and they progressed into Additional Tasks. The number of final trials per individual ranged from four to eight (mean = 4.5).

Because of scheduling constraints, Phase II began two weeks after Phase I was completed.

Before the start of the additional tasks, Raccoons 29 and 40 were given a “refresh” trial, where they were presented with the same set up as their final trials. Both raccoons immediately began

4 dropping stones into the tube and retrieved the reward, and we therefore began Phase II additional tasks.

Size Task

All stones used in the size task were black in color and only varied slightly in shape and weight (small: mean = 17.11g, SE = 1.09g; large: mean = 63.3g, SE = 2.97g). One large stone was required to raise the water level to a point where the reward was within reach, whereas all three small stones were required to achieve the same amount of water displacement (see ESM

Figure 1a for an image of the six stones). We predicted that if the raccoons understood the principles of water displacement and the functionality of the stones, they should preferentially use large stones and thus be more efficient in obtaining the reward.

Substrate task

The water-filled apparatus contained 500mL water, and the corncob-filled apparatus contained corncob litter measured to the same height as the water tube (see ESM Figure 1b for an image of both apparatuses). The corncob litter (i.e., coarsely-ground pieces of corncob) was routinely used in the raccoons’ litterboxes and was deemed a safer alternative to sand or sawdust, which are typically used in the Aesop’s Fable paradigm (e.g., Cheke et al. 2012; Logan et al.

2014). The raccoons were given the same five stones used in Phase I. Because dropping stones into the apparatus containing corncob litter did not elevate the reward (i.e., was non-functional), we predicted that raccoons would place more stones into the apparatus containing water rather than the apparatus containing corncob.

Density task

All balls were similar in appearance, and only deviated visually from one another by their numbers (see ESM Figure 1c for an image of the six balls used in the density task). The three

5 heavy balls would sink when placed in water (mean weight = 51.1g, SE = 1.08), whereas the three hollow balls were light and would float when dropped in water (mean weight = 16.3g, SE =

1.09). Due to the space the balls occupied in the tube, we cut the marshmallow reward into even smaller pieces for this task. We predicted that if raccoons perceived the difference in the functionality of the sinking versus floating balls, they would preferentially use the sinking balls.

Tool use

The small, steel cup with a handle in the tool use task was a 1/8 cup commercial coffee scooper (see Figure 1d for an image of the cup). If the raccoons dropped the cup into the tube, the cup would float momentarily before sinking to the bottom; this raised the water level only marginally and therefore left the reward inaccessible. Raccoons appeared able to grip the handle of the cup with one forepaw while extending their arms, which furthered the reaching ability of the raccoons. This observation led us to believe that the raccoons would be physically capable of using the cup to scoop marshmallows out of the apparatus. Therefore, we predicted that if raccoons perceived the functionality of the cup, they would use it to retrieve the reward instead of dropping it into the tube.

Statistical Analyses

We used Generalized Linear Models (GLM) with a Poisson distribution (i.e., count data) or beta distribution (i.e., proportion data bounded between 0-1). All models were built using

Program R (R Core Team, 2015). Our model selection was based on Akaike’s Information

Criterion corrected for small sample sizes (AICc) and Akaike weights (package: MuMIn; Bartoń

2016). If a model included >.90 weight, or was the intercept only model, we selected this as our top model. If there was not a clear top model, then we performed model averaging (see ESM

Table 1 for a summary of our model selection).

6 All data was collected from continuous video recordings of trials. We predicted that as raccoons learned to drop stones into the apparatus to retrieve the reward, work time —amount of time they spent interacting with the apparatus and stones— would eventually decrease across trials. We used GLMs with a Poisson distribution (package: lme4; Bates et al. 2014) to determine the extent that work time (response variable) was affected by trial number, subject ID, or an interaction between these fixed effects. We also predicted that as raccoons learned to drop stones into the apparatus, exploratory diversity — the number of mutually-exclusive behaviors expressed towards the apparatus/stones— would change across trials (Benson-Amram and

Holekamp 2012). Specifically, we expected the proportion of useful behaviors, or those behaviors that facilitated success, to increase across trials as raccoons learned the actions necessary for retrieving the reward and the proportion of non-useful behaviors to decrease as raccoons learned how to solve the task (Chow et al. 2016). We categorized all behaviors as useful (e.g., touch tube, stand on platform, pick up stone) or non-useful (e.g., sniff tube, lick platform, wash stone), depending on whether the behavior was required for retrieving the reward

(see ESM Table 2 for our ethogram, which includes categorization of all behaviors). We then used GLMs with a beta distribution to determine if the proportion of useful behaviors (response variable) was affected by trial number, subject ID, or an interaction between these fixed effects

(package: betareg; Cribari-Neto and Zeileis 2010). Similarly, we used GLMs with a beta distribution to assess if the proportion of correct vs. incorrect choices made across trials in Phase

II was affected by trial number, subject ID, or an interaction between these fixed effects

(package: betareg; Cribari-Neto and Zeileis 2010).

7 Inter-observer reliability

Inter-observer reliability of work time and exploratory diversity was calculated for nine randomly selected trials (14% of total trials for successful raccoons) using a Pearson product moment correlation coefficient (r = .99). Inter-observer reliability for choices made by each raccoon (correct, incorrect, no choice) was calculated for 12 randomly selected trials (14% of total choice task trials) using a Cohen’s Kappa (k = 0.95). During the size and density trials, the raccoons dropped multiple stones and objects into the tube at one time. In these instances, we scored which stone or object made contact with the water first.

Additional Details - Results

During the three initial trials, five of the eight raccoons approached the apparatus, and four of these raccoons interacted with the apparatus and/or stones (mean interaction time=184 seconds; range = 0-1067 seconds). All of the raccoons approached the apparatus during at least two of their learning trials, although only three raccoons learned to drop stones into the tube as a result.

Raccoon 29 began independently dropping stones into the apparatus during his seventh learning trial (tenth trial overall), Raccoon 40 began dropping stones during his first final trial (sixteenth trial overall), and Raccoon 22 began dropping stones during her fourth learning trial (seventh trial overall). Both Raccoons 29 and 40 retrieved the reward in all six final trials by dropped stones into the tube. Despite our efforts to build an apparatus that raccoons should not have been able to lift based on data collected from a previous NWRC study (QA-2263), Raccoon 22 overturned the apparatus during her fourth final trial (nineteenth trial overall).

Although Raccoons 29 and 40 were the only raccoons to successfully retrieve the floating reward by dropping stones into the apparatus, we included Raccoon 22 in our Phase I statistical analyses because she did drop stones into the tube and did successfully solve the problem and

8 retrieve the food reward when she overturned the apparatus. The results of our model selection revealed that work time was influenced by an interaction between subject ID and trial number

(see ESM Table 1 and ESM Figure 2). Because Raccoon 22 was able to innovate a novel solution in one trial, we extended our exploratory diversity analysis of useful behaviors to include the behaviors Raccoon 22 exercised when she overturned the apparatus (i.e., move apparatus, pull apparatus; see ESM Table 2). Again, our top model indicated that there was interaction effect of trial number and subject ID on the proportion of useful behaviors expressed

(see ESM Table 1 and ESM Figure 3).

Tool use task

Raccoons 29 and 40 failed to retrieve the reward by gripping the cup and inserting it into the tube. In several trials, however, after dropping the cup into the tube, both raccoons attempted to quickly retrieve the cup before it sank. We observed that this “fishing” behavior could result in the reward being captured in the cup upon the initial drop, and consequently obtained when the raccoon retrieved the cup. Raccoon 29 performed this strategy in Trials 1, 4, 5, 8, 10, and 11, and successfully captured the reward in Trial 11 (note: Trial 9 was excluded because of video file corruption). Raccoon 40 performed this strategy in Trials 2, 4, 8, 10 and 11, and was successful at obtaining the reward in Trial 4. In a minority of trials (one trial for Raccoon 29 and four trials for Raccoon 40) raccoons did not drop the cup into the tube at all, but instead spent the entire trial (approximately 1,200 seconds) interacting with the cup.

Additional Details - Discussion

Unlike the more typical learning pattern demonstrated by Raccoon 29, work time for

Raccoon 40 increased across trials. We believe this is because the stones, which varied in shape, had to be aligned correctly to fall into the opening of the tube. Because our apparatus needed to

9 be tall enough to account for raccoon forearm length, we designed the diameter of the tube to be narrow so that when stones were dropped, the displacement of water would be substantial enough to be noticed by the raccoons. In keeping the opening narrow, however, the stones had to be aligned in a precise way to fall in. Aligning the stones correctly likely required some practice to achieve efficiently, and Raccoon 40 did not start dropping stones independently until his first final trial. Notably, he struggled to get a stone into the tube in his sixth final trial, indicating that although he understood what actions needed to be accomplished (i.e., he was attempting to drop stones into the tube), he was not physically skilled at inserting the stones into the opening of the tube by the end of his trials. In contrast, Raccoon 29 began interacting with the task sooner, and therefore had gained more efficient dropping skills by the start of his final trials. Despite this dissimilarity in work time, both Raccoon 29 and Raccoon 40 demonstrated learning as the proportion of useful behaviors expressed increased over time. Although Raccoon 22 did start dropping stones into the tube, she never demonstrated strong evidence of learning by measure of work time nor proportion of useful behaviors, most likely because her stone-dropping actions were never reinforced by successful retrieval of the reward.

Additional Hypotheses of Consideration

Additional hypotheses have been presented to explain the correct discrimination seen in choice tasks excluding causal understanding, such as the “object bias” hypothesis (i.e., previous experience working with a water apparatus or stones/other heavy objects may predispose animals to selecting similar materials when given a choice) and the “perceptual feedback" hypothesis

(i.e., successful animals are simply observing what actions are bringing their reward closer and are repeating those actions) (Taylor et al. 2010; Taylor et al. 2012; Jelbert et al. 2015). Other studies suggest that subjects in Aesop’s Fable experiments employ trial-and-error learning as

10 opposed to causal understanding to solve this paradigm (Ghirlanda and Lind 2017), or perhaps a combination of multiple cognitive strategies (Taylor et al. 2011). Our small sample size limited our ability to conduct a more in-depth investigation of such alternative hypotheses; however, our data do suggest that a bias for heavy objects is not likely driving the response of raccoons in this task. Had the raccoons been inclined to use the larger stones and/or the sinking objects, this could have indicated a bias based on weight. But because we did not observe a preference for heavier stones and objects in the large vs. small stones or sinking vs. floating objects tasks, it is unlikely the raccoons formed such a bias.

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