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Discrimination of predatory versus nonpredatory mammals by box turtles,Terrapene carolina

Roy King, Robert Gosnell and Alicia Mathis

Department of Biology, Missouri State University, Springfield, Missouri 65897, USA

Summary. Although terrestrial turtles have served as amodel for studies of olfactory neurophysiology, little isknown about how they use chemical information in anecological sense. We tested whether box turtles (Terra-pene carolina) use chemical information to distinguishbetween predatory and nonpredatory mammals. Boxturtles in our study exhibited more escape behavior whenexposed to urine from a predator (coyote, Canis latrans)than when exposed to urine from a nonpredator (white-tailed deer, Odocoileus virginianus) or a blank control.Escape behavior is consistent with an antipredator re-sponse. In addition, the turtles decreased their handlingtime for food when in the presence of urine from eitherspecies of mammal in comparison to the blank, indicatingthat chemical cues frommammals in generalmay result inincreased vigilance by terrestrial turtles. Examination of avariety of response variables may be important for ade-quate assessment of the ecological role of chemosensorybehavior.

Key words. Antipredator behavior – chemical cues –chemosensory behavior – Terrapene carolina – turtle

Introduction

For vertebrates, box turtles (Terrapene sp.) often serve asa model for the study of chemosensory neurophysiology(e.g., Tonosaki 1993; Wachowiak & Cohen 1999; Lam etal. 2000;Wachowiak et al. 2002). In contrast to the wealthof mechanistic information about the olfactory system ofbox turtles, little is known about the ecological function ofbox turtle olfaction. An exhaustive review of box turtleecology by Dodd (2002) listed only two early, relativelyqualitative studies of responses to olfactory cues, whichinvolved food recognition (Allard 1949; Fitch 1965). In

box turtles, the fright center of the brain, the amygdala,receives fibers from the olfactory tract (Carey 1970),suggesting that another function of olfaction may be indetection of cues related to predation.

The ability to assess the danger associated with pre-dators and to adjust behavior accordingly can have apositive effect on survival of prey (Azevedo-Ramos etal. 1992; Downes 2002). Antipredator responses, such asflight or hiding, are more likely to be effective when theyoccur early in a predation sequence (Lima & Dill 1990).Detection of predators through olfactory cues can allowprey to detect predators before they become visible. Thischaracteristic of chemosensory cues may be particularlyimportant for animals such as terrestrial turtles that oc-cupy habitats where vision is often obstructed by tallgrasses or other vegetation.

Among the reptiles, olfactory detection of predatorshas been best studied in the squamates (e.g., lizards:Cooper 1990; Phillips & Alberts 1992; Downes & Shine1998; snakes: Weldon et al. 1992; Miller & Gutzke 1999).The potential for chemical detection of predators byturtles has received little attention, and we are aware ofno other studies of chemosensory detection of predatorsby terrestrial turtles.

Three-toed box turtles, Terrapene carolina triunguis,inhabit mainly woodlands (Schwartz & Schwartz 1974),but also use open grasslands and prairies during parts ofthe year (Reagan 1974; Sammartano 1994). Althoughthey can be locally abundant, box turtles are listed onAppendix II of CITES, meaning that they have been af-forded amoderate level of international protection. Mostthreats to the survival of the species are related to habitatdestruction, over-collection by humans for the pet trade,and highway mortality (Dodd 2002). The most seriousnatural threats include predation by canids (Canis sp.),raccoons (Procyon lotor), skunks (Mephitis mephitis),and other predators (Auffenberg & Iverson 1979; Dodd2002). Due to their abundance (Missouri Department ofConservation 1981; Kamler 1998) and size, canids, in-Correspondence to:Alicia Mathis, email: AliciaMathis@missouristate.

edu

Chemoecology 18: 61 – 64 (2008)0937-7409/08/010061-4ABirkhBuser Verlag, Basel, 2007DOI 10.1007/s00049-007-0393-9

CHEMOECOLOGY

cluding coyotes (Canis latrans), are probably a particu-larly important threat to adult box turtles.

We tested whether three-toed box turtles can dis-criminate between a blank control and urine from pre-datory (coyote) and nonpredatory (white-tailed deer,Odocoileus virginianus) mammals. Both species arecommon in box turtle habitats (personal observations),and so should be familiar to our wild-caught turtles. Wequantified two types of response variables: (1) Time inEdge behavior (snout or forelegs pushing against the sideof the testing chamber) and (2) Foraging activity (Latencyto attack and Handling Time [time from first strike toconsumption]). We follow other studies in interpretingEdge behavior as an antipredator response (e.g., Mathis& Lancaster 1998; Hickerson et al. 2004) because it isconsistent with attempted flight or attempts to find hidingplaces. Decreases in activity, including foraging, often areassociated with fright responses (e.g., Kieffer 1991; An-gradi 1992). Typically, decreased activity occurs eitherdue to an overall increase in the rate of vigilance (Mag-nhagan 1988) or when decreased movements lead tolower rates of detection by visual predators (Azevado-Ramos et al. 1992).

Methods

We obtained 18 three-toed box turtles (Terrpene carolina triunguis)from theNiawathe Prairie ConservationArea located 12.9 km north ofLockwood, in Dade County, Missouri. The turtles were captured in thespring and summer of 2004 and over-wintered in the laboratory atMissouri State University in Greene County, Missouri. While in thelaboratory, the turtles were kept in groups in stainless steel tanksmeasuring (in cm) either 176 I 74 I 31 or 120 I 23.5 I 31. A mixture ofsoil and moistened hardwood mulch was used for bedding. Tank tem-peratures were maintained between 24 and 288 C, and full-spectrumUV lighting (12:12 hr light:dark cycle) was used to ensure turtle health.Turtles were fed earthworms (Lumbricus terrestris) or red and greenleaf lettuce 2—3 times per week and were given fresh water every 1—2days as needed. To standardize hunger levels and to insure that theturtles were “hungry” during the trials, turtles were given only lettucefor approximately two weeks before trials and were not fed at all forfour days before testing.

The predator scent was coyote urine (100%) obtained from Ad-irondack Outdoor Co, Elizabethtown, NY, and the nonpredator scentwas Doe-in-Estrus urine (100%) obtained from a local retailer. Wechose to use urine as the stimulus because urine is deposited by somecoyotes on substrates as territorial markers (Bowen & Cowen 1980;Gese&Ruff 1997), and so turtles are likely to encounter thesemarkersduring their daily activities. Moreover, because the territorial spacingsystem indicates a high degree of site specificity, the presence of thesemarkers is likely to indicate a high probability that a coyote is in thearea. Plain tap-water was used as a “blank” control. Earthworms wereused as the food resource during testing. Each turtle was tested in eachof the three treatments (only once per treatment), with the order oftesting and assignment of treatments random for each round of trials.The repeated measures designed allowed us to have a relatively robustsample size per treatment without requiring collection of additionalturtles. Turtles were maintained in individual 42.5-L opaque (blue)plastic containers during the testing period.

Each trial was conducted in a 17-L clear plastic box that had beenplaced inside a cardboard box to block the turtleOs view of their sur-roundings. Between trials, testing containers were rinsed with ex-tremely hot water, dried and then rinsed with isopropyl alcohol. Whitecotton balls (25 I 23 mm) were saturated with coyote or deer urine or

water andwere kept in closed sterile bottles (5.8 I 7.2 cm) prior to trialsto minimize escape of air-borne cues into the testing room. Duringtrials, the bottle with the cotton ball was opened and placed in a cornerat one end of the testing chamber, and a single worm was placed in theother corner at the same end of the chamber. The scent of the urine wasimmediately detectable by the human observers. The test turtle wasthen placed at the opposite end of the chamber. Trials were endedwhenthe turtle completely consumed the worm or after 7 min.

We quantified Latency and Handling Time of feeding and percenttime in Edge behavior using stopwatches. Data for Latency and Han-dling Time were analyzed using repeatedmeasures ANOVAs followedby Tukey multiple comparison tests. Turtles that did not consume awormreceived a score of 7 min forLatency andwere not included in theanalyses of Handling Time. Because data for Edge behavior were notnormally distributed, these data were analyzed with a nonparametricFriedmanOs Test. Pair-wise comparisons of Edge treatments were madewith Wilcoxon matched-pairs signed-ranks tests with a Bonferronicorrection to alpha (a=0.025) because each data set was used morethan once. Analyses were made with the Minitab statistical software,release 14. Experimental turtles were released at the place of collectionafter completion of this study.

Results and Discussion

Percent time in Edge behavior was significantly differentamong treatment groups (FriedmanOs test: S=12.06,P=0.002, n=18; Fig. 1).According toWilcoxon post-hoctests, turtles spent significantly more time in Edge in thecoyote treatment than both the blank (W=45,P=0.0039) and deer (W=1, P=0.0039) treatments, butthere was no significant difference between the blank anddeer treatments (W=15, P=0.9).

The response to the coyote scent (increased edge be-havior) is consistent with a predicted antipredator re-sponse because, in nature, this behavior would lead theturtle away from the area or into potential hiding places.Escape behavior as a response to predatory cues has beenreported for most major taxa (e.g., invertebrates: Hemmi

Fig. 1 Time spent in Edge behavior (mean + 1SE) for box turtles ex-posed to chemical stimuli (urine) from coyote (predator), white-taileddeer (nonpredator) and a blank control. Coyote was significantly dif-ferent from the other treatments..

62 R. King, R. Gosnell and A. Mathis CHEMOECOLOGY

2005; fishes: Brown & Warburton 1999; amphibians:Mathis & Lancaster 1998; reptiles: Blazquez et al. 1997;birds: Blumstein 2003; mammals: Bonenfant & Kramer1996). Fright responses to urine of predatory mammalshas been reported for numerous species of small mam-mals (e.g. , Epple et al. 1993; Nolte et al. 1994; Rosell2001), but studies of chemoreception of predators byreptiles typically have used nonmammalian predators(Weldon 1990). We are aware of only one other study ofpredator recognition via chemoreception in turtles, inwhich aquatic musk turtles, Sternotherus carinatus and S.minor, avoided areas marked by predatory alligatorsnapping turtles,Macroclemys temmincki (Jackson 1990).

Regardless of treatment, turtles were fairly slow toinitiate foraging bouts (repeated measures ANOVA:F=0.08, P=0.9, n=18; Fig. 2a). Time to consume prey(Handling Time) was different among treatments(ANOVA: F=4.14, P=0.03, n=8; Fig. 2b), with turtlestaking a longer time to consume prey in the blank treat-ment than in the deer treatment (TukeyOs test: T=–2.8,P=0.026) but not in the coyote treatment (TukeyOs test:T=–1.8, P=0.18). Handling Time did not differ signifi-cantly between the coyote and deer treatments (TukeyOstest: T=0.996, P=0.59).

The foraging data in our study should be interpretedwith caution because less than half (44% in each of thetreatments) of turtles consumed prey during the study.However, for the turtles that fed, time to consume prey(handling time) was significantly influenced by treat-ment, with turtles taking the longest time to consumepreyin the blank treatment. Because shorter handling timescan lead to the availability of more time for vigilancebehavior (Lima&Dill 1990), decreased handling times inthe two urine treatments may indicate that the turtleswere at least somewhat disturbed by the presence of thedeer scent as well as the coyote scent. Interestingly, thelack of a significant difference between the predator andnonpredator treatments cannot be explained by failure ofthe turtles to recognize the difference between the twoscents; discrimination ability clearlywas demonstrated bythe differences in escape behavior. The presence of pre-dator/nonpredator scents did not significantly affectfeeding latency.

The results differed depending on which of the threeresponse variables (edge, latency, handling time) wereanalyzed. These differences underscore the importanceof examining multiple response variables in behavioralassays of predator recognition. If we had only recordedforaging responses, we would have reached an erroneousconclusion concerning ability of turtles to discriminatebetween cues from predatory and nonpredatory mam-mals.

Ability to distinguish between predatory and non-predatory odors may have important fitness conse-quences because unnecessary responses to species thatare not dangerous can result in loss of foraging or re-productive opportunities (Lima&Dill 1990). The relativecomplexity of the turtle olfactory system, as indicated by

neurophysiological studies (e.g., Lam et al. 2000), appar-ently has led to a high degree of specificity in thechemosensory discrimination abilities of box turtles. Se-lection for specificity in responses to odors from potentialpredators may be widespread. For example, wall lizards(Podarcismuralis) can distinguish between substrate cuesfrom predatory and nonpredatory snakes (Amo etal. 2004), graybelly salamanders (Eurycea tynerensis,formerly E. multiplicata griseogaster) can distinguish be-tween aquatic cues from predatory and nonpredatoryfishes (Hickman et al. 2004), and red-bellied tamarins(Saguinus labiatus) can distinguish between fecal scentsof predatory and nonpredatory mammals (Caine &Weldon 1989).

Acknowledgements

We thank D. Moll for allowing us to use his laboratoryspace and L. Gosnell for her assistance with data collec-tion. Turtles were collected under the authority of Mis-souri Department of Conservation collecting permit#12267.

Fig. 2 Time spent performing two aspects of foraging behavior forturtles exposed to chemical stimuli from coyote, white-tailed deer and ablank control. (a) Latency to strike at an earthworm and (b) HandlingTime (time from strike to consumption). Treatments with the sameletter did not differ significantly; data are presented as mean + 1 SE.

Discrimination of predatory versus nonpredatory mammals by box turtles, Terrapene carolinaVol. 18, 2008 63

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Received 5 July 2007; accepted 20 October 2007Published Online First 23 November 2007

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