For Peer Review Journal: Zoo Biology

Zoo Biology

The Flehmen Response and Pseudosuckling in a Captive, Juvenile Southern Sea Otter (Enhydra lutris nereis)

Manuscript ID Draft

Wiley - Manuscript type: Research Article

Date Submitted by the Author: n/a

Complete List of Authors: Island, Heide; Pacific University, Psychology Wengeler, Julia; Pacific University, Psychology Claussenius-Kalman, Hannah ; Pacific University, Psychology

Keywords: flehmen, pheromone, sea otter, olfaction, stereotypy

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Island, 1 Sea Otter Flehmen 1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 The Flehmen Response and Pseudosuckling in a Captive, Juvenile Southern Sea Otter 18 For Peer Review 19 20 (Enhydra lutris nereis) 21 22 Heide D. Island* 23 24 Julia Wengeler 25 26 Hannah Claussenius-Kalman 27 28 Department of Psychology, Pacific University, Forest Grove, Oregon 29 30 Manuscript Word Count: 4,589 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 *Correspondence to: Heide Island, Department of Psychology, Pacific University, 2043 College 55 56 57 Way, Forest Grove, OR 97116; Email: [email protected]; Phone: 503-352-1538 58 59 60 John Wiley & Sons Zoo Biology Page 2 of 37

Island, 2 Sea Otter Flehmen 1 2 3 4 Abstract 5 6 7 A juvenile, female sea otter (Enhydra lutris nereis) was observed in 43 instances of the flehmen 8 9 response over 19 days from May through July of 2015 at the Oregon Zoo. In all flehmen grimace 10 11 observations, the juvenile sea otter engaged in nibbling, nosing, or licking the peri-mammary or 12 13 anogenital areas of a non-lactating, geriatric female sea otter. The stereotypic flehmen behavior 14 15 observed was consisted with the sequences of behavior documented in other mammals, lifting the 16 17 head, elevating the nose to the air, retracting the upper lip slightly, and manipulating her mystacial 18 For Peer Review 19 20 vibrissae back and forth while rapidly inspiring air through her mouth in quick succession, tongue 21 22 extruded. The occurrence of this behavior was not specific to visitor density, visitor impact rating, 23 24 day of the month, time of day, or exhibit zone. However, it did occur more frequently in one area 25 26 of the enclosure. Among the three sea otters (two females, one male) currently housed at the Oregon 27 28 Zoo, the juvenile female’s flehmen response only occurred following interactions with the older 29 30 female and was always preceded by the pseudosuckling or anogenital nosing, licking or nibbling 31 32 33 behavior. 34 35 36 37 Key words: flehmen, pheromone, sea otter, olfaction, stereotypy 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 3 of 37 Zoo Biology

Island, 3 Sea Otter Flehmen 1 2 3 4 INTRODUCTION 5 6 7 The flehmen is a behavioral sequence that involves the elevation of the head, retraction of 8 9 the upper lip, and the inhalation of air through the mouth (Estes, 1972). Depending upon the 10 11 species, the tongue may be extended beyond the lips and curled (Poddar-Sarkar and Lal 12 13 Brahmachary, 2014), or pushed against the hard palate (Hart, 1982). Further, the flehmen may be 14 15 accompanied by an oral aspiration (Charpentier, et al 2013), wrinkling of the nose (Weeks, et al. 16 17 2002), or partial closing of the nares (Bailey, 1978). The apparent function of the flehmen grimace 18 For Peer Review 19 20 is to sip the air for semiochemicals, heavy molecules that communicate reproductive status (Estes, 21 22 1972; Sankar and Archunan, 2004), reproductive synchrony (Thompson, 1995), social dominance 23 24 (Thompson, 1991), territory (Begg, et al 2003), maternal behavior (Aron, 1979; Wysocki, 1972) or 25 26 conspecific identification (Swaisgood, et al 1999). The putative pheromones sampled through the 27 28 flehmen are often suspended in biological secretions from anal or sternal glands, vaginal discharge, 29 30 urine, feces, milk, saliva, or other biological fluid (For a thorough review of mammalian 31 32 33 semiochemicals, see Ferrero and Liberles, 2009). Through the flehmen response, air is pulled 34 35 across the tongue into the back of the throat and along the orifices of the vomeronasal organ (VNO) 36 37 (Pageat and Gaultier, 2003). 38 39 The VNO is the chemoreceptive, peripheral sensory organ of the accessory olfactory 40 41 system, thought to be anatomically and physiologically distinct from the main olfactory system 42 43 44 (MOS). Most tetrapod vertebrates possess a vomeronasal organ regardless of their reliance on 45 46 chemosensensory communication, with the exception of cetaceans, sirenians, old world primates, 47 48 and bats (Mackay-Sim, et al. 1985). The VNO consists of two mucus-filled tubes on either side of 49 50 the inferior nasal septum, where olfactory receptor neurons (ORN) line the medial region of the 51 52 VNO epithelium (Hart 1982). Even though the main olfaction system appears to have some 53 54 pheromone receptivity (Kelliher, 2007; Yoon, et al. 2005; Zufall and Munger, 2001) current 55 56 57 wisdom of mammalian pheromone reception posits that the vomeronasal system (VNS) is the most 58 59 60 John Wiley & Sons Zoo Biology Page 4 of 37

Island, 4 Sea Otter Flehmen 1 2 3 4 specialized for semiochemical detection (Buck, 2000; Yu, et al. 2010). During the flehmen 5 6 7 response, semiochemicals are inhaled through the mouth and mix with mucous, passing into the 8 9 VNO triggering chemosensory impulses from VNO olfactory receptor neurons to the accessory 10 11 olfactory bulb (Ladewig and Hart, 1982). Thus the flehmen grimace heightens chemosensation 12 13 among those vertebrates that utilize intraspecific pheromone communication. 14 15 First documented among ungulates and felids (Schneider, 1930 as cited in Bailey, 1978), 16 17 the flehmen response has been observed across broad taxonomic genera. Among ungulates, goats 18 For Peer Review 19 20 (Ladewig and Hart, 1980; Melese-D’Hospital and Hart, 1985), antelope (e.g., Frey, et al. 2007; 21 22 Thompson, 1991; 1995), bovines (Houpt, et al. 1988; Sankar and Archunan, 2004), horses (e.g., 23 24 Christensen, et al 2005; Weeks, et al 2002), tapirs (Tortato, et al 2007), rhinoceroses (Zahari, et al. 25 26 2005), hippopotamuses (Zapico, 1999), and giraffes (Bercovitch, et al 2006) utilize the flehmen 27 28 during social communication. Similarly, the flehmen is widely used within the felid family for 29 30 intraspecific chemoreception of social state, examples include: the domestic cat (Hart and Leedy, 31 32 33 1987), tigers (Poddar-Sarkar and Brahmachary, 2014), panthers (McBride and McBride, 2010), 34 35 leopards (Freeman, 1982), and lions (Powell, 1995). The flehmen response was documented among 36 37 several other terrestrial mammals including pandas (Swaisgood et al 2002), elephants (Rasmussen 38 39 and Greenwood, 2003; Rasmussen et al 2002), lemurs (Bailey, 1977; Evans,1980), and mandrills 40 41 (Charpentier et al 2013). Although domestic dogs (Canidae) and ferrets (Mustelidae) are not 42 43 44 thought to engage in a true flehmen response, other members within their families, like the maned 45 46 wolf (Canidae) (Coelho et al 2012) and the honey badger (Mustelidae) do (Begg, et al 2003). 47 48 In spite of our understanding of olfactory and chemosensory perception among terrestrial mammals, 49 50 the use of olfaction and chemosensation by semiaquatic marine mammals is poorly understood. 51 52 While fully aquatic mammals, like the cetaceans and the sirenians have either reduced olfactory 53 54 tracts or absent olfactory bulbs (Berta, et al, 2006; Mackay-Sim et al, 1985; Yu, et al, 2010) relying 55 56 57 primarily on the sensory modalities of audition and vision; there is evidence for the use of olfaction 58 59 60 John Wiley & Sons Page 5 of 37 Zoo Biology

Island, 5 Sea Otter Flehmen 1 2 3 4 in social interaction among the seals, sea lions, and walruses (Dobson and Jouventin, 2003; Miller, 5 6 7 2002). In fact, given the existence of a vomeronasal system, the large size of the ethmoturbinal 8 9 bones, and their ability to modulate the diameter and the opening of their nares, (Sergeant, 1991), 10 11 pinnipeds are thought to have a sharp sense of smell (Schusterman, 1981; Sergeant, 1991; Yu et al, 12 13 2010). 14 15 Harp seal mother and pups have been observed in face-to-face “nosing” behavior, which 16 17 may be a form of scent marking since the sebaceous glands below the eye secrete an oil that retains 18 For Peer Review 19 20 a unique odor (Sergeant, 1991). The acceptance of pups by their mothers using the mutual naso- 21 22 nasal contact has also been observed in fur seals (Phillips, 2003; Dobson and Jouventin, 2003). 23 24 Further, sniffing the facial regions between mother and pup may aid in detecting secretions from the 25 26 sweat glands, located between the mystacial vibrissae, thereby reinforcing the maternal bond (King, 27 28 1991; Mackay-Sim et al. 1985). In the event the mother and pup separate, Wyatt (2014) observed 29 30 cow harp seals facing the wind to use airborne scent trails of the pup to recover their young. During 31 32 33 mating season, sea lions as well as some species of seals (e.g., harp seals and ringed seals) emit a 34 35 strong odor during rut, likely serving to identify territory during male-male competition (Sergeant, 36 37 1991). Moreover, both male and female pinnipeds nose anogenital and perineal regions during 38 39 breeding and pupping season (Sergeant, 1991). There is even documentation of flehmen-like facial 40 41 grimacing and tongue extrusion among sea lions following social displays (Miller, 2002). Fur seals 42 43 44 in a food-rewarded, two-choice, instrumental conditioning task were able to discriminate between 45 46 five different classes of aliphatic odorants, chemicals associated with high marine productivity 47 48 (Laska et al. 2010). Laska and colleagues (2010) demonstrated that of 25 different chemical 49 50 concentrations and compositions, fur seals could discriminate between 24 of them. In an 51 52 experimental study of olfactory sensitivity among gray seals, Kowalewsky and colleagues (2006) 53 54 presented differing concentrations of dimethyl sulphide (DMS), a reliable atmospheric odor of 55 56 57 productive foraging areas, using an operant go/no go response method. The seals were able to detect 58 59 60 John Wiley & Sons Zoo Biology Page 6 of 37

Island, 6 Sea Otter Flehmen 1 2 3 4 the presence of DMS in over 85% of all presented concentrations (Kowalewsky et al, 2006). DMS 5 6 7 is also one of fifteen organosulfur compounds identified in scent marking secretions of mustelids, 8 9 including otters. This suggests that otters may also be sensitive to DMS in olfactory cues (Burger, 10 11 2005; Yu et al, 2010). These studies, taken together indicate that olfaction may play a more critical 12 13 role in behavior regulation among semiaquatic marine mammals than previously thought. 14 15 Despite their adaptability to a wide variety of habitats (e.g., fresh water, coast marine, 16 17 18 estuarine, and riparian)For, climates, andPeer range, the anatomy Review and physiology of otter olfaction is poorly 19 20 understood (Berta et al, 2006). Like their mustelid cousin, the honey badger (Begg, et al 2003), 21 22 river otters engage in chemocommunication by expelling organosulfur compounds in scat (i.e., 23 24 spraints), scat jellies (mucous deposits), urine, or anal gland secretions (Gorman, et al. 1978; 25 26 Oldham and Black, 2009) throughout their range. These spraint deposits or latrines, appear to 27 28 delineate territory (Kruuk 1992) and convey social status (Rostain, et al 2004). Yet there is no 29 30 31 published documentation of flehmen behavior in wild or captive settings for any of the 13 extant 32 33 species of otters. To our knowledge, the olfactory morphology and the existence of the VNO 34 35 among otters is also unexplored in the literature. In a genetic analysis of the TRPC2 gene, one of 36 37 three vomeronasal system (VNS) genes, Yu and colleagues (2010) isolated and characterized the 38 39 TRPC2 gene among four species of marine mammals, the California sea lion, harbor seal, fin whale 40 41 and river otter. Since the TRPC2 gene is frequently used as a genetic marker for VNO pheromone 42 43 44 signaling, their study attempted to broaden the current understanding of pheromone communication 45 46 relative to mammals’ evolutionary transition from land to water. Yu and colleagues (2010) found 47 48 the river otter, sea lion, and harbor seal shared 94% sequence identity of the TRPC2 gene even 49 50 though the TRPC2 sequence of the harbor seal and river otter were pseudogenes. This suggests that 51 52 the semiochemicals associated with scent marking, characteristic of river otters, may be perceived 53 54 by one of the other two VNS-specific genes, like the vomeronasal receptor type 1 (V1R) or 2 55 56 57 (V2R), or by the main olfactory system rather than the vomeronasal system. 58 59 60 John Wiley & Sons Page 7 of 37 Zoo Biology

Island, 7 Sea Otter Flehmen 1 2 3 4 Sea otters are less understood anatomically than pinnipeds, or even among other mustelids, 5 6 7 yet their neuroanatomy (England and Dillon, 1972), gustatory and olfactory apparatuses are 8 9 analogous to their terrestrial relatives with the exception of slightly reduced olfactory bulbs (Lowell 10 11 and Flanigan, 1980). Although sea otters unlike other mustelids, have no anal scent glands 12 13 (Dudzinski, et al. 2002), sea otter social behavior implies a more central role in olfaction (Berta, 14 15 2006; Estes, 1989). Through remote field observation, Estes (1989) reported sea otters scenting the 16 17 presence of humans by moving down-wind, particularly in areas where sea otters are less 18 For Peer Review 19 20 accustomed to human odors. Similarly, Winn and Schneider (1978) reviewed rafting sites of sea 21 22 otter beds and posited that persistent sheltered, upwind locations indicated a reliance on olfaction 23 24 for predator detection. During breeding season, male sea otters were observed sniffing the air to 25 26 identify nearby females (Riedman and Estes, 1990), prior to copulation anogenital nosing was also 27 28 29 common, presumably to scent semiochemicals indicative of estrous (Estes, 1989). Comparable to 30 31 nose-to-nose rubbing and scent marking between seals, sea otter sow and pup pairs also reportedly 32 33 engage in both facial and anogenital nosing and grooming (Hanson et al. 1993). When encountering 34 35 one another in the water, sea otters scent genitalia in a manner equivalent to intraspecific 36 37 semiochemical discrimination among other carnivores (Lowell and Flanigan, 1980). 38 39 Based on the limited anatomical literature of mustelids and the comparative olfactory 40 41 42 sensitivity of pinnipeds, the sea otter should have a well-developed sense of smell and may also 43 44 engage in chemosensory behavioral regulation. In fact, in an unpublished study of sea otter 45 46 olfactory sensitivity, a male Northern sea otter and a Southern sea otter were tested independently at 47 48 the Oregon Coast Aquarium and the Oregon Zoo respectively using yes/no olfactogram 49 50 presentations of seven odorant compounds (Hammock, 2005). The two sea otters’ olfactory 51 52 response accuracy was between 72 and 89 percent (Hammock, 2005). Despite Hammock’s results, 53 54 55 indicating sea otter olfactory sensitivity of some odorant compounds within the range of terrestrial 56 57 58 59 60 John Wiley & Sons Zoo Biology Page 8 of 37

Island, 8 Sea Otter Flehmen 1 2 3 4 carnivores, there is virtually no published literature on olfaction or chemoreceptive behavior among 5 6 7 any of the lutrine species. 8 9 MATERIALS AND METHOD 10 11 Focal Subjects 12 13 The subjects of this research were three captive Southern sea otters in the Steller Cove 14 15 exhibit at the Oregon Zoo. The Oregon Zoo adopted Eddie, (male, age 17, approximately 68 16 17 pounds; International Species Information System Number: A00049) and Thelma (female, age 17, 18 For Peer Review 19 20 approximately 48 pounds; International Species Information System Number: A00050) from 21 22 Monterey Bay Aquarium after they were found as orphaned pups in 1998 (Oregon Zoo, 2014). 23 24 Eddie was one of two subjects in the only sea otter olfactory discrimination study (Hammock, 2005) 25 26 described in the introduction. Unlike Eddie, whose growth was far below the normal growth curve 27 28 for sea otter pups prohibiting rerelease back into the wild, the Oregon Zoo made multiple attempts 29 30 to return Thelma to a natural environment. Yet due to her reliance and comfort around humans, 31 32 33 Thelma did not demonstrate competent foraging skills to thrive in a natural marine ecosystem. In 34 35 2005, several years after the successful birth of a healthy male pup, both Thelma and Eddie were 36 37 prescribed a form of birth control called Deslorelin. However, in 2012 Eddie was castrated and 38 39 Deslorelin was no longer administered to either sea otter. Certain health issues in the elders could 40 41 affect behavioral patterns: Eddie is blind in his left eye and Thelma lost her left lung due to 42 43 44 pneumothorax. The youngest sea otter, Juno (female, age 1.7 years, approximately 46 pounds, 45 46 International Species Information System Number: B40094) was found at approximately 4-weeks 47 48 old orphaned along Cayucos State Beach in California. Juno joined the Oregon Zoo through their 49 50 surrogate-rearing program with the hope that Thelma, given her calm temperament would foster 51 52 Juno. During the four years Oz, Thelma and Eddie’s offspring, lived at the Oregon Zoo (Oz was 53 54 relocated to the Georgia Zoo in 2005), Thelma demonstrated attentive pup-rearing skills. Thelma 55 56 57 58 59 60 John Wiley & Sons Page 9 of 37 Zoo Biology

Island, 9 Sea Otter Flehmen 1 2 3 4 and Juno immediately bonded and several weeks after their successful surrogacy Eddie and Juno 5 6 7 were introduced (DeGroot, 2013; DeGroot, 2015). 8 9 Materials 10 11 Observations were digitally video recorded using a Sony FDRAX33 4K digital video 12 13 camcorder mounted on a 12-foot telescoping Sunpak Ultra tripod. Two observers manually 14 15 recorded behavior using a behavioral ethogram printed on legal sized Rite in the Rain® all weather 16 17 copy paper and a 11”x17” acrylic, side-fastener clipboard. Finally, all manually recorded data was 18 For Peer Review 19 20 transcribed into SPSS® statistical software using a MacBook Pro® 10.10.3 laptop, digital data was 21 22 archived into an online cloud, using DropBox® and Quicktime®. 23 24 Observations 25 26 Procedure. The focal naturalistic observation was conducted over 10 weeks, with 2 27 28 researchers recording data 3 hours per day, 4 days per week (120 hours), from May through July 29 30 2015. Focal observations consisted of 10 minute blocks divided into 30-second intervals. 31 32 33 Periodically, researchers stopped between the 10-minute observation intervals to focus the camera 34 35 or zoom in on a specific specific behavior. The observers recorded data for the same otter in order 36 37 to track inter-observer agreement. Generally, when multiple sea otters were on exhibit, their 38 39 behaviors overlapped so selecting a focal animal in advance was not typically necessary. For times 40 41 in which multiple animals were on exhibit and engaged in autonomous activity, both recorded 42 43 44 behavior for a single focal animal and then referred to the video footage to record behavior for the 45 46 other animals in the laboratory. The interobserver agreement was calculated using Cohen’s kappa 47 48 (N=1,895 observations) for the two observers over the 10-week period, yielding a kappa value of 49 50 .85, indicating substantial agreement. 51 52 Ethogram. Since behavior is community-specific, based on the training, experience, age, 53 54 sex, and cohesiveness of a given community, each community will have their own specific 55 56 57 behavioral repertoire. The establishment of a valid ethogram requires a pilot period of just 58 59 60 John Wiley & Sons Zoo Biology Page 10 of 37

Island, 10 Sea Otter Flehmen 1 2 3 4 observing the culture of a given community and the dynamics of the members within that 5 6 7 community. Some animals only engage in certain behavior in the presence of another animal or in 8 9 the absence of people, or with fewer people, or when the noise level is high, etc. These kinds of 10 11 complex behavioral nuances cannot be ascertained without simply observing. The initial pilot 12 13 period for the two observers was one week, prior to formal data collection. The pilot ethogram was 14 15 was based on Packard and Ribic’s (1982) paper classifying sea otter behavior and activity budget. 16 17 Each day resulted in the exclusion of unobserved behaviors and the inclusion of specific behaviors 18 For Peer Review 19 20 repeatedly evidenced by the sea otters at the Oregon Zoo. The ethogram that emerged from this 21 22 initial pilot period included three behavioral categories: Resting and Grooming; Locomotion and 23 24 Eating; and Socializing and Play and seven contextual categories: Time of Day; Zone Region; Zone 25 26 Side; Visitor Density; Visitor Impact (VI) rating; Sea Otter(s) on Exhibit; and Presence of 27 28 Abnormal Repetitive Behavior. Those behaviors within the Resting and Grooming category 29 30 include: Raft, Float, Sleep, Self-stimulation (i.e., licking at the anogenital areas or for Eddie, his 31 32 33 penis), Chinning, Pseudosuckle (a sequence of behavior that include Juno biting, licking, nosing, or 34 35 suckling the periteat or abdominal areas on Thelma), Anogenital (nosing or grooming of the 36 37 anogenital area in another animal or on themselves), Flehmen (this behavior was added following 38 39 the pilot period as it repeatedly emerged during observation), Logroll, Dunk, Half Roll, Somersault, 40 41 Tuckroll, Stroke, Rub, and Bite/Lick (for a description for those behaviors without explanation, see 42 43 44 Packard and Rubric, 1982). The behavioral category of Locomotion and Eating included the 45 46 following fifteen behaviors: Periscope, Dive, Underwater Swim, Swim, Porpoise, Side Stroke, 47 48 Submerge, Sculling, Forage, Pound, Food (i.e., food handling), Eat, Chew (this occasionally 49 50 included biting on or chewing the enclosure substrate), Land (climb onto terrestrial areas of the 51 52 exhibit), and Walk. The last thirteen behaviors are within the domain of Socialization and Play, 53 54 these included: Toy, Shake, Blows Bubbles, Nip, Copy, Chase, Headlock (holding the head while 55 56 57 wrestling), Take, Give, Lunge, Nose (the nose is directed in a solicitous behavior toward another 58 59 60 John Wiley & Sons Page 11 of 37 Zoo Biology

Island, 11 Sea Otter Flehmen 1 2 3 4 otter), Paw, and Ride. For all unexplained behaviors, see Packard and Rubric (1982) for detailed 5 6 7 behavioral narratives. 8 9 Visitor Variables. We used two visitor variables to help identify contextual cues in which 10 11 visitors may contribute to dramatic changes in otter behavior or abrupt otter relocation to another 12 13 area of the enclosure. The first visitor variable, Visitor Density was simply the total number 14 15 visitors by zone. The second variable, the Visitor Impact Score, required the observers to assign a 16 17 quantitative rating of how loud, invasive, or just generally disruptive the visitors were while 18 For Peer Review 19 20 observing the sea otters. The Visitor Impact (VI) rating was assigned using a continuous scoring 21 22 system of 1 (i.e., the presence of a human visitor or observer) to 10 (i.e., the animal was displaced 23 24 by their behavior or forced into an interaction) to identify the level of disruption or behavioral 25 26 change the visitors’ presence caused among the sea otters on exhibit. For example, a Low Impact 27 28 rating of 1-3, indicated that the observers did not influence the animals’ behavior beyond visual 29 30 contact or curiosity. Low Moderate Impact was rated as a VI rating of 4 or 5; High Moderate 31 32 33 Impact was a VI rating of 6–7 and High Impact was a score of 8–10, where 10 was direct 34 35 interaction with the otter. A high score meant that while in the presence of the visitor, the animal 36 37 changed their behavior in some significant way (e.g., moved to another zone) in order to either 38 39 avoid or engage with the visitor. An example of a Visitor Impact Score of 10 occurred when a 40 41 woman dropped her bracelet into the enclosure while leaning over the railing at the South 42 43 44 Observation window. Eddie dove for the bracelet, retrieved it and proceeded to pound the bracelet 45 46 on the habitat. The observers notified the Zoo staff and within several minutes, keepers coaxed 47 48 Eddie off exhibit and recovered the visitor’s bracelet. 49 50 Enclosure Zones. In order to record where in the habitat behaviors occurred and the 51 52 location of visitors when referencing visitor variables, the exhibit was divided in to six zones. The 53 54 zones were numbered clockwise from Zone 1, at the south observation window (Figure 1). Zones 2 55 56 57 58 59 60 John Wiley & Sons Zoo Biology Page 12 of 37

Island, 12 Sea Otter Flehmen 1 2 3 4 through 4 and 6 reflected features or areas of the exhibit visible from Zone 1, south observation 5 6 7 window or Zone 5, the north observation window (Figures 2 and 3). 8 9 RESULTS 10 11 This account of a sea otter flehmen response was derived from direct observation and 12 13 digital video footage archived during 10 weeks of focal observation. The original project goal was 14 15 to document three sea otters’ activity budget, enrichment interaction, and habitat utilization at the 16 17 Oregon Zoo. Juno, at the time of observation, a 19-month old juvenile, female sea otter evidenced 18 For Peer Review 19 20 all flehmen behaviors outlined here. Her flehmen or flehmen-like grimace always followed 21 22 contextual suckling, or “pseudosuckling” with Thelma, at the time of observation, a 17-year old, 23 24 postbred non-nursing female (Table 1). The documented pseudosuckling-flehmen behavioral 25 26 sequence occurred over the course of 19 days from May through July of 2015. Both the observers 27 28 and the zoo staff were aware of the pseudosuckling behavior prior to our documentation. Thelma 29 30 received maintenance care for small lesions around the areas of her nipples, presumably due to 31 32 33 Juno’s licking or pseudosuckling (DeGroot, Personal Communication, March 29, 2015). 34 35 The pseudosuckling-flehmen behavioral sequence emerged during resting or rafting 36 37 between Thelma and Juno and was generally predicated by Juno “chinning” or placing her chin on 38 39 the chest or abdomen of Thelma. The was followed by Juno rooting with her nose through 40 41 Thelma’s pelage (Figure 4) and suckling, nibbling or licking Thelma’s teat or periteat area (Figure 42 43 44 5). In isolation, the duration of the pseudosuckling behavior varied from approximately 10 seconds 45 46 to several minutes. When coupled with the flehmen, Juno lifted her head from Thelma’s abdomen, 47 48 elevated her nose to the air, retracted her upper lip slightly, manipulated her mystacial vibrissae 49 50 back and forth while rapidly inspiring air through her mouth in quick succession, tongue extruded 51 52 (Figure 6). The pseudosuckling behavior did occur in the absence of a flehmen response but the 53 54 flehmen did not occur in the absence of pseudosuckling. Though it did also follow anogenital 55 56 57 nosing as well. The typical duration of the flehmen was between 10 to 20 seconds, wherein Juno 58 59 60 John Wiley & Sons Page 13 of 37 Zoo Biology

Island, 13 Sea Otter Flehmen 1 2 3 4 either resumed the contextual suckling or engaged in a new behavior. 5 6 7 In an effort to ascertain if visitor variables mediated the pseudosuckling or flehmen 8 9 behavior we conducted a Pearson’s correlation with pseudosuckling, flehmen, visitor density and 10 11 visitor impact ratings. Indeed, visitor variables did influence the pseudosuckling behavior, as it was 12 13 significantly negative correlated with both visitor number, r(14,597) =-.035, p <.001 and visitor 14 15 impact ratings, r(14,615) =-.021, p=.01. In other words, pseudosuckling was more frequent during 16 17 low visitor number and low visitor impact ratings. While the flehmen behavior was not 18 For Peer Review 19 20 significantly correlated with any of the visitor variables. The pseudosuckling-flehmen behavioral 21 22 sequence occurred across the 3-month observation period, without necessarily following monthly or 23 24 diurnal pattern. A one-way analysis of variance revealed that while the behavioral sequence 25 26 occurred across zones, it was more frequent in Zone 5 at the North Observation window (F (2, 27 28 11,257) = 24.21, p<.001, !2=.013) than the other five zones, but the effect size was small. 29 30 31 Ethics Guidelines Acknowledgement. The authors abide by Wiley's ethics guidelines and 32 33 by the guidelines produced by the Committee on Publication Ethics. Although this study was 34 35 submitted to Pacific University’s Institutional Animal Care and Use Committee (IACUC) for 36 37 consideration, it was deemed “Exempt” from review given the study posed no additional risks or 38 39 harm to the observational animals than daily zoo visitor interaction. In other words, the project was 40 41 entirely behavioral observation with no direct interaction with or manipulation of the sea otters 42 43 44 observed at the Oregon Zoo. 45 46 DISCUSSION 47 48 This behavioral account provides to our knowledge, the first documentation of flehmen or 49 50 flehmen-like behavior among any of the lutrine species. The conditions in which Juno 51 52 demonstrated the flehmen response occurred during episodes of pseudosuckling, abdominal licking, 53 54 or anogenital exploration, and only with Thelma. Juno’s flehmen behavioral sequence included an 55 56 57 elevated head, retracted lips, slightly extended tongue, and inspirations of air through the mouth, 58 59 60 John Wiley & Sons Zoo Biology Page 14 of 37

Island, 14 Sea Otter Flehmen 1 2 3 4 consistent with flehmen behavior and contextual triggers that evoke the flehmen grimace in other 5 6 7 species (Thompson 1995; Tortato et al. 2007, Weeks et al. 2002). Since no hormonal assays were 8 9 evaluated in conjunction with this behavioral study, it is difficult to know what, if any, 10 11 semiochemical prompted Juno’s flehmen behavior. Since Thelma was not nursing and thereby 12 13 provided no nutritional support, this might explain Juno’s exploratory attempts of chemoreception 14 15 through the flehmen response. The emergence of Juno’s flehmen or flehmen-like behavior may 16 17 reflect an attempt to identify through olfaction Thelma’s hormonal state, particularly if the behavior 18 For Peer Review 19 20 was an attempt at nursing rather than simply bonding or grooming behavior. 21 22 There is the possibility that the flehmen sequence is stereotypic behavior since it could be 23 24 argued that Juno’s persistent pseudosuckling or licking, which predicated the flehmen, is consistent 25 26 with abnormal repetitive licking (Shyne, 1989). Shepherdson (1989) asserted that stereotypes, 27 28 repetitive, invariant behavior with no immediate function, frequently emerge when an animal has 29 30 prolonged exposure to an ecologically relevant problem and is constrained by the captive context. 31 32 33 Given the strength of Juno and Thelma’s attachment, and the initial timing of Juno’s rescue, pre- 34 35 weaned, this may explain both her early attachment and suckling. Thereby the failure to nurse 36 37 generalized the suckling behavior to abdominal or anogenital nosing and licking. Morabito and 38 39 Bashaw (2012) reported in a survey of 61 river otter exhibits and 55 institutions with a total sample 40 41 of 129 captive North American River otters, 46% of the otters engaged in ARBs. Though pacing 42 43 44 was the most commonly reported abnormal repetitive behavior, one otter engaged in persistent 45 46 extrusion of its tongue, though the contextual circumstances of this were unreported. Therefore, it is 47 48 possible that rather than an ARB, the one case of river otter repetitive tongue extrusion may have 49 50 been a misidentified example of flehmen or flehmen-like behavior. 51 52 We would argue Juno’s flehmen does not reflect stereotypic behavior. If we consider the 53 54 typical stressors in a captive environment which may contribute to an abnormal or stereotypic 55 56 57 repetitive behavior, they include: 1.) boredom from limited enrichment or an inability to perform 58 59 60 John Wiley & Sons Page 15 of 37 Zoo Biology

Island, 15 Sea Otter Flehmen 1 2 3 4 specifies-typical feeding and foraging behavior; 2.) high visitor density or disruptive noise; 3.) non- 5 6 7 natural habitat, a restricted range within the habitat, or absence of retreat space; and 4.) disrupted 8 9 intraspecific cohesion (Morgan and Tromborg, 2007). Regarding the first stressor, limited 10 11 enrichment and an inability to engage in species-typical feeding and foraging behavior, the Oregon 12 13 Zoo incorporates enrichment activities and training in all physical therapies, husbandry, and 14 15 veterinary care (DeGroot, personal communication, March 29, 2014). Additionally, more than 200 16 17 enrichment items are rotated both on and off exhibit to facilitate problem-solving and sea otter- 18 For Peer Review 19 20 typical foraging behavior (DeGroot, personal communication, March 29, 2014). In addition, 21 22 creative enrichment activities have been incorporated into physical therapy as well. For example, 23 24 Eddie was taught to dunk balls into a basketball hoop so he could exercise his arthritic elbows. To 25 26 the second contextual contributor of ARBS, high visitor density and environmental noise may stress 27 28 captive animals (Mason, 2010). Based on Pearson’s correlations with the flehmen and visitor 29 30 variables, the emergence of the flehmen was unrelated to visitor density or visitor impact rating. 31 32 33 Even with respect to the sequence of Juno’s pseudosuckling, these are typical of resting behavior 34 35 and consequently were more likely to occur during times of low visitor density and lower visitor 36 37 impact ratings. With respect to the third challenge, pertaining to of habitat constraints, the Stellar 38 39 Cove Sea Otter enclosure affords exhibited animals shade and direct sun, terrestrial substrate above 40 41 water, water depth commensurate with diving and exploring underwater, natural features (e.g., 42 43 44 trees, foliage, moss, boulders, rock outcroppings) to explore above and below water, retreat areas 45 46 (e.g., Zone 4), as well as an observation window for curious animals to actively engage with 47 48 visitors. Juno and Thelma demonstrated only a marginal habitat preference for the pseudosuckling- 49 50 flehmen behavioral sequence, Zone 5 at the South Observation Window, one area in which Juno 51 52 and Thelma engaged in the pseudosuckling-flehmen behavioral sequence, Zone 5 at the South 53 54 Observation window, as this behavior was observed in all six zones of the habitat. Finally 55 56 57 regarding issues of social cohesion, the keepers are able to adjust which animals are on exhibit, if 58 59 60 John Wiley & Sons Zoo Biology Page 16 of 37

Island, 16 Sea Otter Flehmen 1 2 3 4 the social harmony is disrupted, the dynamic can be re-established by moving animals off exhibit. 5 6 7 The standard accepted definition of stereotypic behavior identifies more than 10% of the waking 8 9 hours devoted to the abnormal, persistent behavior and for Juno in the 40 days of observation over 10 11 the 10-week period, fewer than half include flehmen events and each event lasted no more than 10 12 13 to 20 seconds, although the pseudosuckling sequence of behavior on some occasions lasted for an 14 15 hour or more. 16 17 In light of the strength of the affiliative bond between Juno and Thelma, the context in 18 For Peer Review 19 20 which the flehmen occurred, the generalizability of Juno’s flehmen response to those of other 21 22 species, and the apparent olfactory function associated with the behavior, we believe it does not 23 24 represent a stereotypic behavior. That said, this represents a purely descriptive study, which of 25 26 course constrains the conclusiveness of these observations. Further investigation of chemoreceptive 27 28 behavior among mustelids or lutrine species might include olfactometry to better confirm the 29 30 flehmen in conjunction with semiochemical or olfactory exploration. 31 32 33 CONCLUSIONS 34 35 1. This study reflects the first documented behavioral account of the flehmen response 36 37 among any lutrine species. 38 39 2. The pseudosuckling-flehmen behavior was negatively correlated with both visitor 40 41 number and visitor impact rating, suggesting the behavioral sequence is more likely a bonding and 42 43 44 resting behavior than a stress-related behavior. 45 46 3. Given Thelma’s advanced age, even among captive sea otters and the fact she had not 47 48 birthed young for well over a decade, no nutritional support was provided during suckling episode, 49 50 it is clear both animals garner some kind of comfort or bonding from this behavior. 51 52 4. The authors acknowledge the possibility the flehmen behaviors observed were an 53 54 extension of the suckling behavior or a novel example of stereotypy from a captive sea otter. 55 56 57 58 59 60 John Wiley & Sons Page 17 of 37 Zoo Biology

Island, 17 Sea Otter Flehmen 1 2 3 4 ACKNOWLEDGEMENTS 5 6 7 The authors would like to the Oregon Zoo and specifically, Karen Lewis, David Shepherdson, 8 9 Alison Heimowitz, and Jenny DeGroot for their willingness to share sea otter biographical data and 10 11 exhibit information as well as their support of this project. 12 13 14 15 CONFLICT OF INTEREST STATEMENT 16 17 The authors have no conflict of interest to report, we are not employees of the Oregon Zoo, nor 18 For Peer Review 19 20 do we have any other potential sources of conflict of interest relevant to the publication of this 21 22 paper, including no patents or stock ownership relevant to the study, membership or service as 23 24 board of directors to the Oregon Zoo, membership of an advisory board or committee for the 25 26 Oregon Zoo, and no consultancy for or receipt of speaker’s fees from the Oregon Zoo or any other 27 28 company relevant to zoo research. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Zoo Biology Page 18 of 37

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Island, 24 Sea Otter Flehmen 1 2 3 4 TABLE 1. 5 6 Location, date, and time of flehmen in a captive female southern sea otter at the Oregon Zoo 7 Date Time Location Preceding Flehmen Behavior Concurrent Flehmen 8 5-21-2015 12:23 Zone 5 Pseudosuckle, Raft, Anogenital Stomach curl 9 10 5-22-2015 10:21 Zone 5 Pseudosuckle, Raft Raft 11 5-22-2015 10:32 Zone 5 Pseudosuckle, Raft, Visitor Disruption Raft 12 5-22-2015 10:55 Zone 2 Pseudosuckle, Raft Scull 13 5-25-2015 10:44 Zone 5 Pseudosuckle, Raft Raft 14 5-25-2015 11:00 Zone 5 Pseudosuckle, Raft, Visitor Disruption Raft 15 5-29-2015 10:47 Zone 2 Pseudosuckle, Raft Raft 16 5-29-2015 11:09 Zone 5 Pseudosuckle, Raft Raft 17 5-29-2015 12:25 Zone 5 Pseudosuckle, Raft Raft 18 5-29-2015 12:40 ForZone 5 PeerPseudosuckle, RaftReview Raft 19 20 6-2-2015 10:44 Zone 3 Pseudosuckle, Raft, Visitor Disruption Raft 21 6-2-2015 11:18 Zone 5 Pseudosuckle, Raft Raft 22 6-4-2015 13:44 Zone 5 Pseudosuckle, Raft Raft 23 6-11-2015 10:58 Zone 5 Pseudosuckle, Raft Scull, Swim 24 6-11-2015 14:13 Zone 1 Pseudosuckle, Raft Scull, Float 25 6-15-2015 14:01 Zone 5 Pseudosuckle, Raft, Anogenital Raft 26 6-16-2015 11:13 Zone 6 Pseudosuckle, Raft Raft 27 6-16-2015 11:24 Zone 5 Pseudosuckle, Raft Raft 28 6-19-2015 9:30 Zone 2 Pseudosuckle, Raft Raft 29 6-19-2015 9:51 Zone 2 Pseudosuckle, Raft Raft 30 7-6-2015 12:38 Zone 5 Pseudosuckle, Raft Raft 31 7-9-2015 14:09 Zone 5 Pseudosuckle, Swim, Drag (Thelma) Raft 32 33 7-10-2015 11:03 Zone 5 Pseudosuckle, Raft Scull 34 7-10-2015 11:14 Zone 5 Pseudosuckle, Raft Scull 35 7-10-2015 12:20 Zone 5 Pseudosuckle, Raft, Anogenital Raft 36 7-10-2015 12:26 Zone 5 Pseudosuckle, Raft, Anogenital Raft 37 7-13-2015 11:19 Zone 5 Pseudosuckle, Raft, Displace (Eddie) Raft 38 7-13-2015 12:35 Zone 6 Pseudosuckle, Anogenital Periscope 39 7-16-2015 12:14 Zone 5 Pseudosuckle, Raft, Anogenital Float 40 7-16-2015 12:28 Zone 5 Pseudosuckle, Raft, Anogenital Float 41 7-23-2015 12:35 Zone 3 Pseudosuckle, Raft Scull 42 7-23-2015 14:01 Zone 3 Pseudosuckle, Raft Scull 43 7-23-2015 14:26 Zone 5 Pseudosuckle, Raft Swim 44 7-29-2015 11:02 Zone 5 Pseudosuckle, Raft, Displace (Eddie) Scull 45 46 7-29-2015 11:14 Zone 4 Pseudosuckle, Raft Scull 47 7-29-2015 11:25 Zone 5 Pseudosuckle, Raft Swim 48 7-29-2015 12:36 Zone 5 Pseudosuckle, Raft Scull, Swim 49 7-30-2015 10:19 Zone 5 Pseudosuckle, Raft Rafting 50 7-30-2015 10:40 Zone 3 Pseudosuckle, Raft, Anogenital Float 51 7-30-2015 10:58 Zone 5 Pseudosuckle, Raft, Anogenital Scull 52 7-30-2015 11:06 Zone 5 Pseudosuckle, Raft Scull 53 7-31-2015 11:23 Zone 3 Pseudosuckle, Float Float 54 7-31-2015 12:14 Zone 5 Pseudosuckle, Periscope Float 55 56 57 58 59 60 John Wiley & Sons Page 25 of 37 Zoo Biology

Island, 25 Sea Otter Flehmen 1 2 3 4 N=43 occurrences over 19 days; “pseudosuckling” reflects a dyad between Juno (suckling) and 5 6 Thelma (target), “Displace” refers to Eddie interacting with the dyad, causing Juno and Thelma to 7 split up and thereby interrupting their behavior. 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29

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Island, 26 Sea Otter Flehmen 1 2 3 4 FIGURE 1. 5 6 7 South Observation Window (171”x40”) 8 9 10 11 12 13 14 15 16 17