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Predator Avoidance in Lab-Reared Juvenile Rough-SkinnedNewts, Taricha granulosaAuthor(s): Jory Johnson Brian G Gall Edmund D Brodie, JrSource: Northwestern Naturalist, 94(2):103-109. 2013.Published By: Society for Northwestern Vertebrate BiologyDOI: http://dx.doi.org/10.1898/12-20.1URL: http://www.bioone.org/doi/full/10.1898/12-20.1

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PREDATOR AVOIDANCE IN LAB-REARED JUVENILEROUGH-SKINNED NEWTS, TARICHA GRANULOSA

JORY JOHNSON

Utah State University, 5305 Old Main HL, Logan UT 84322; jo.johnson@aggiemail.usu.edu

BRIAN G GALL

Hanover College, PO Box 108, Hanover IN 47243

EDMUND D BRODIE, JR

Utah State University, 5305 Old Main HL, Logan UT 84322

ABSTRACT—Predator avoidance strategies are often viewed in the context of innate or learned,yet a true test of this hypothesis requires animals that are completely naı̈ve to potential predators.We examined the predator avoidance behavior of juvenile Rough-skinned Newts (Tarichagranulosa) that had been reared in captivity since being deposited as eggs and had never beenexposed to predators or predator stimuli. In contrast to a previous study on adult newts, juvenilesavoided a broader range of chemical stimuli from potential predators, including alarm cues fromdamaged conspecifics and stimuli from 2 Common Gartersnakes (Thamnophis sirtalis) that hadrecently consumed newts. These results suggest that predator avoidance in Taricha granulosa isinnate. Unlike adult newts, the avoidance of a wider range of stimuli by juvenile newts is likely aneffective strategy at reducing predation risk given their small size and lower tetrodotoxinconcentrations (compared to adults), both of which render them vulnerable to predation bygartersnakes.

Key words: alarm cues, innate, juvenile, naı̈ve, predator avoidance, Rough-skinned Newts,Taricha granulosa, tetrodotoxin, TTX

The ability of prey to recognize and respondto predators is vital for their survival (Lima andDill 1990). Prey possess multiple sensory sys-tems that aid them in recognizing when apredator is nearby. For example, visual, tactile,and auditory cues are important for manyorganisms, depending on the particular habitatin which they reside (Endler 1992). Chemicalcues are often used by prey to detect when apredator is in the area (Kats and Dill 1998;Wisenden 2003; Ferrari and others 2010). Typesof chemical cues that are perceived by preyinclude kairomones (the scent of a predatoralone), alarm cues from injured individuals (achemical emitted by injured prey that commu-nicates to conspecifics that danger is imminent),and cues due to dietary factors such as thoseemitted after a predator has eaten a prey item(see review in Ferrari and others 2010; Wisen-den 2003). Once prey have identified a predator,they may use a variety of avoidance and

antipredator behaviors including immobility,taking shelter, fleeing, or defensive posturing(Brodie 1983; Endler 1992; Kats and Dill 1998).

Many studies have tested the response ofanimals to chemical stimuli from potentialpredators (see review in Ferrari and others2010). However, a prey animal’s response tochemical stimuli, including alarm cues andkairomones, is dependent on the conditionsthe animal was exposed to throughout its life.For example, prey can learn to avoid predatorsbased on experience from previous exposure tothat predator (Miller and others 1990; Mathisand Smith 1993; Wisenden 2003), or fromwatching other conspecifics respond withavoidance to a specific predator or predatorstimulus (Griffin 2004; Ferrari and others2007a). Although this is how many organismsavoid predators, some organisms possess theability to recognize and respond to predationrisk with no prior experience (for example,

NORTHWESTERN NATURALIST 94:103–109 AUTUMN 2013

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Vilhunen and Hirvonen 2003). In these cases,predator recognition is innate. Brodie andothers (1991) theorized that in order for a preyanimal to exhibit innate avoidance responses toa particular predator, the prey needs to have co-evolved with the predator over evolutionarytime. This is likely to be facilitated in environ-ments and under predation regimes that arestable over evolutionary time (Ferrari andothers 2007b). Documenting innate predatorrecognition is often difficult, however, becausemany organisms are capable of learning as soonas they are born, and some species are evencapable of learning to avoid predators as anembryo prior to hatching (Mathis and others2008). Demonstrating innate predator recogni-tion, therefore, requires focal animals that arecompletely naı̈ve and reared in captivity fromthe earliest possible stage (egg deposition).

In one of the most well documented coevo-lutionary systems, newts of the genus Tarichaare prey to gartersnakes (Thamnophis spp.)(Brodie and Brodie 1990; Brodie and others2005; Feldman and others 2009). Newts possessthe neurotoxin tetrodotoxin (TTX) in their skin(Wakely and others 1966; Hanifin and others1999, 2008), which functions by blocking volt-age-gated sodium channels in nerves andskeletal muscle, resulting in asphyxiation anddeath (Kao 1966; Narahashi and others 1967).Tetrodotoxin is extremely potent (500 timesmore lethal than cyanide; Mosher and others1964), and successfully deters almost all poten-tial predators of newts (Brodie 1968). Newttoxicity is variable among populations (Hanifinand others 1999, 2008) and is likely the result ofpredation by TTX-resistant snakes (Brodie andBrodie 1990; Brodie and others 2002). Thesesnakes have evolved resistance to the toxin viaamino-acid substitutions to the sodium channelprotein, and some populations can consumemany newts without being harmed (Brodie andothers 2002; Geffeney and others 2002, 2005;Feldman and others 2009, 2010).

The population of Taricha granulosa from SoapCreek Ponds, Benton County, Oregon has a verythe high toxicity level (up to 28 mg of TTX) andhas only a single successful predator, Thamno-phis sirtalis (Common Gartersnake) (Brodie1968). This population is particularly wellstudied, and previous research on predatorrecognition by adult Rough-skinned Newts in

this population has demonstrated that theyrespond with avoidance to stimuli from snakesthat had recently eaten a newt (Gall and others2011a). However, newts did not respond toeither snake kairomones or alarm cues frommacerated newt skin, suggesting that a learnedcomponent may be involved in snake predatorrecognition for adult newts (Gall and others2011a). The authors hypothesized that due tothe newts extreme toxicity, avoidance of allsnake cues was unnecessary because only asubset of individuals in the predator snakepopulation are capable of consuming an adultnewt (Gall and others 2011a). Juvenile newts aresubstantially less toxic than adults (Gall andothers 2011b) and can likely be consumed by allthe snakes in the sympatric predator popula-tion, given the range of resistance to TTX(Brodie and others 2002). This may favoravoidance responses toward all snake cuesrather than only those that have consumedconspecifics. Because Rough-skinned Newtshave co-evolved with their predator, the Com-mon Gartersnake, naı̈ve newts may have devel-oped an innate avoidance response to garter-snakes over evolutionary time.

In this study we exposed juvenile Rough-skinned Newts that were lab-reared from eggdeposition and which have never been exposedto a predator (naı̈ve) or chemical stimuli frompotential predators, to 4 different stimuli: snake-fed-newt chemical cues, snake cues, maceratednewt skin, and a blank control. The goal of thisstudy was to determine whether juvenileRough-skinned Newts exhibit innate avoidanceresponses to predatory stimuli.

METHODS

Animal Collection and Maintenance

Juvenile Rough-skinned Newts were rearedfrom eggs deposited in the lab by gravid femalenewts collected in 2010 from Soap Creek pondsin Benton County, Oregon. A description ofadult, egg, and larval newt husbandry can befound in Gall and others (2011b). Juvenile newtswere housed in groups of 2 to 7 in 2 L containersthat were lined with a damp paper towel andpartially filled with sphagnum moss. Juvenileswere fed bloodworms in shallow dishes 5times/wk prior to testing. Crickets were dustedwith calcium and vitamin dust and provided

104 NORTHWESTERN NATURALIST 94(2)

every 2 to 4 wk. All juveniles were approxi-mately 1-y post-metamorphosis at testing.

The snakes used in this experiment were 2long-term captive Common Gartersnakes thatwere highly resistant to tetrodotoxin (TTX).These snakes also served as the stimulus sourcein a previous study that examined the responseof adult newts to snake cues (Gall and others2011a). Snakes were housed individually in 75-Laquaria lined with newspaper that included ahide box full of damp sphagnum moss as wellas a water dish. Snakes were fed mice weekly,except for when the snake-fed-newt stimuluswas collected.

Stimulus Collection

The juvenile newts were exposed to one of 4treatments: snakes that had not recently con-sumed a newt (n 5 27); snakes that had been feda newt (n 5 27); macerated newt skin (n 5 27);or filtered tap water [blank control (n 5 28)].The snake-only stimulus was collected from the2 snakes 3 d after feeding on a mouse duringtheir normal feeding schedule. Food was thenwithheld for 1 wk and both snakes were offeredlive newts for 2 consecutive weeks. The snake-fed-newt stimulus was collected 3 d after the2nd newt was consumed. One of the stimulussnakes did not consume a newt (this snake hadeagerly consumed live newts in previousexperiments, and continued to feed normallyon mice after these trials); therefore the snake-fed-newt stimulus was based on stimulus froma single snake.

Each stimulus set was obtained by placing 1snake in a 3.7-L glass jar. Thirty-five ml offiltered tap water was added to the jar and a lidwith 4 holes was then screwed onto the jar toprevent escape. After 24 h the snake wasremoved, its mass was recorded, and 5 ml tapwater/g body mass was then added to the jar.The original 35 ml of water was subtracted fromthe total volume of water to be added to the jarto ensure that the stimulus was diluted withexactly 5 ml water/g body mass. Stimulussolutions from the different snakes within atreatment were combined, mixed, and frozen at-806 C in 50-ml tubes in 7-ml aliquots. The blankcontrol was treated the same except no snakewas added to the jar. The macerated newt skintreatment was obtained by removing the skin(2.8 g) from 2 previously frozen newts from the

same locality as the parents of the juvenilestested and macerating it with a mortar andpestle (Hanifin and others 2004). It was thenmixed with 800 ml of filtered tap water andfrozen in 7-ml aliquots. All stimulus vials werecoded prior to testing to ensure the observerswere blind to the treatment.

Test Chamber

The test chamber consisted of a 9.46-L opaquebucket. The floor of the chamber was lined with2 semi-circular paper towels separated by a1.5-cm gap to prevent cross-contamination ofstimuli. One side of the chamber was marked‘‘A’’ and the other side was marked ‘‘B.’’ One ofthe sides was randomly chosen to be treatedwith 7 ml of stimulus solution while the otherside was treated with 7 ml of the previouslyfrozen blank filtered tap water. A clear plastictube (9 cm dia. 3 12 cm long) was set on end inthe middle of the test chamber. The chamberwas misted 3 times with filtered tap water toincrease humidity and standardize the moisturelevel on both sides of the paper towels. Juvenilenewts were randomly placed inside the cleartube in the test chamber for a 20 min acclima-tion period. To maintain moisture levels duringthe trials, and to reduce external visual influ-ences, we placed plastic wrap over the top of thetest chamber and covered it with an opaque lidthat had a small hole for observation. After theacclimation period, the cylinder was removedand the position of the newt’s head (A or B) wasrecorded every 5 min for 2 h. Because of thenewts’ small size, they were able to climb ontothe sides of the test chamber. When all 4 limbswere off of the bottom of the chamber, werecorded the position of the juvenile (A or B)and that the individual was on the wall. Inorder to prevent side bias, the chamber wasrotated 906 every 30 min; this technique iscommonly used to control for side bias inexperiments exposing terrestrial salamandersto chemical cues from predators (Cupp 1994;Lutterschmidt and others 1994; Marvin andHutchison 1995; Chivers and others 2001; Galland others 2011a). The stimulus vials werecoded prior to experimentation and the treat-ments were applied by a 2nd observer who wasnot involved in data collection; therefore, theidentity and position of the treatment wasunknown to the primary observer. One animal

AUTUMN 2013 JOHNSON AND OTHERS: PREDATOR AVOIDANCE IN JUVENILE NEWTS 105

was removed from the analysis because it didnot move from the center of the container.Between trials the buckets and acclimationcylinders were cleaned with hot water anddried with paper towels. A total of 110 juvenilenewts were tested. Newts were tested in only 1treatment and were never retested.

The numbers of observations on the treat-ment and blank sides of the test chamber weresummed for each trial. Wall observations wererecorded as avoidance of the stimulus. Wecompared the number of observations on thetreatment and control sides of the test chamberfor each stimulus type using a Paired t-test, ora Wilcoxon signed rank test for data that didnot meet assumptions for parametric statistics.This or similar analyses are commonly used toevaluate the responses of terrestrial amphibi-ans to chemical stimuli (Cupp 1994; Luttersch-midt and others 1994; Marvin and Hutchison1995; Chivers and others 2001; Gall and others2011a). We compared the mean number of wallobservations between the different stimuliwith an ANOVA on ranks due to non-normality.

RESULTS

Juvenile newts were observed less often onpaper towel that had stimuli from snakes thathad consumed newts (Z 5 2.42, df 5 26, P 5

0.016, Fig. 1), as well as on areas of paper towelthat had stimuli from macerated newt skin (t 5

22.48, df 5 26, P 5 0.02, Fig. 1). Juvenile newtsalso tended to avoid paper towel that hadsnake-only stimuli (Z 5 1.64, df 5 26, P 5 0.104,Fig. 1). When exposed to paper towels withcontrol stimuli, juvenile newts spent similaramounts of time on each side of the testchamber (t 5 21.11, df 5 27, P 5 0.278,Fig. 1). We found a significant difference inthe number of wall observations between the 4treatments (H 5 9.19, df 5 3, P 5 0.027, Fig. 2),with the fewest number of wall observations inthe control treatment and the most in the snake-fed-newt and newt-skin treatments (Fig. 2).

DISCUSSION

The juvenile newts that we used in thisexperiment were lab-reared and were naı̈ve toany contact with predators or predator odors.

FIGURE 1. Mean (±SE) proportion of time naı̈ve juvenile Rough-skinned Newts (Taricha granulosa) spent onthe stimulus side of the test container when exposed to one of 4 treatments including: Control 5 filtered tapwater (n 5 28); Snake 5 filtered water with chemical cues from Thamnophis sirtalis that had not consumed a newt(n 5 27); Snake fed newt 5 filtered water with chemical cues from T. sirtalis that had recently consumed a newt(n 5 27); and Newt skin 5 filtered water with macerated newt skin (n 5 27). Dashed line indicates equaldistribution between both sides of the test chamber. Asterisk signifies P , 0.02 compared to random.

106 NORTHWESTERN NATURALIST 94(2)

This suggests that any response the juvenilesexhibited toward chemical stimuli from thepotential predators was not learned fromexperience, but was instead innate and has agenetic component. Our results show thatjuvenile Rough-skinned Newts respond withavoidance to alarm cues released by injuredconspecifics, as well as to kairomones from theirmajor predator, gartersnakes, that had recentlyeaten newts. We interpreted the wall climbingbehavior observed in these trials as the juvenilenewts attempting to move away from thestimulus. This is a behavior not reported inadult newts from the same locality (Gall andothers 2011a) and might be explained as theinability of the adults, with a greater mass, toclimb the side of the chamber.

In a similar experiment that tested naı̈vesalamanders, Eurycea nana (San Marcos Sala-mander), it was found that the predator-naı̈vesalamanders avoided the native and non-nativepredator stimuli while the predator-experiencedsalamanders only avoided the native predatorstimulus (Epp and Gabor 2008). These resultsdemonstrated that E. nana have an innaterecognition and response to some predator

stimuli (Epp and Gabor 2008). Similarly, Galland Mathis (2010) demonstrated that naı̈velarval Hellbenders (Cryptobranchus alleganiensis)have an innate antipredator response towardnative fish predators.

In an experiment exposing adult Rough-skinned Newts (from the same population usedin our experiment) to snake stimuli, the newtsresponded by avoiding only chemical cues fromsnakes that had consumed a newt (Gall andothers 2011a). In comparison to the adultresponse, juveniles avoided a greater diversityof stimuli. Previous studies have demonstratedthat naı̈ve animals may exhibit less specificantipredator responses compared to experi-enced adults. For example, naı̈ve juvenileViviparous Lizards (Lacerta vivipara) do notbask on a substrate with predatory snakestimuli, whereas adult lizards will bask in thepresence of these cues (Damme and others1995), although the authors also suggest thatthese results could have been caused by age-related variation in thermal preferences or byheating rate. The trend observed in our study ofjuvenile newts avoiding a range of predator-related stimuli will likely diminish as they

FIGURE 2. Mean (±SE) number of observations of naı̈ve juvenile Rough-skinned Newts (Taricha granulosa) onthe wall of the test container when exposed to one of 4 treatments including: Control 5 filtered tap water; Snake5 filtered water with chemical cues from Thamnophis sirtalis that had not consumed a newt; Snake fed newt 5

filtered water with chemical cues from T. sirtalis that had recently consumed a newt; and Newt skin 5 filteredwater with macerated newt skin. H [3] 5 9.19, P , 0.027.

AUTUMN 2013 JOHNSON AND OTHERS: PREDATOR AVOIDANCE IN JUVENILE NEWTS 107

grow, develop more TTX, and acquire experi-ence with predators.

Organisms must balance trade-offs betweenpredator avoidance and fitness-enhancing ac-tivities such as foraging and reproduction (Limaand Dill 1990). Although juvenile newts avoid-ed a broad range of predatory chemical stimuli,the slow developmental rate and delayed sexualmaturity of newts suggests that juvenile newtsshould exhibit a strategy that minimizes theirchances of death. Although juvenile newtslikely expend more energy avoiding predatorscompared to the adults, their innate ability torecognize and avoid predators should enhancethe probability of survival because a dangerousinitial encounter with a predator is not requiredto learn which stimuli to avoid. Nevertheless,this extra expenditure of energy should onlylast until the juveniles develop enough TTX todeter predators.

The probability of prey exhibiting innatepredator avoidance responses is likely depen-dent on the stability of the predation regime theprey is exposed to as well as the coevolutionaryhistory between a specific predator and prey(Brodie and others 1991; Ferrari and others2007b). Newts from the Soap Creek populationused in this study are at the center of one of thestrongest coevoluationary hotspots betweensnakes and newts (Brodie and others 2002;Hanifin and others 2008), and newt toxicity isclosely matched with snake resistance in thispopulation (Hanifin and others 2008). Thisrelationship, in addition to the reduction in thenumber of predators capable of consumingnewts due to the presence of TTX, likelyfacilitated the genetic fixation of the antipreda-tor responses toward snakes.

We demonstrated that juvenile Rough-skinned Newts were able to detect and avoidalarm cues from conspecifics as well as alarmsignals in conjunction with snake stimuli.Because the juveniles we used had not beenexposed to any snake predator or odor before,they were completely naı̈ve and their responsewas innate.

ACKNOWLEDGMENTS

We thank L Hoffmann for help with animal care

and data collection. This research was conducted

under Utah State University’s animal use protocol

#1008R. Funding was provided by Utah State Univer-

sity Biology Department and the USU College ofScience.

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