RESEARCH ARTICLE The behavioural effects of predator-induced stress responses in the cricket (Gryllus texensis): the upside of the stress response

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1 468 The Journl of Experimentl Biology 216, Pulished y The Compny of Biologists Ltd doi:1.1242/je RESEARCH ARTICLE The ehviourl effects of predtor-induced stress responses in the cricket (Gryllus texensis): the upside of the stress response Shelley A. Admo*, Ily Kovlko nd Brinn Mosher Deprtment of Psychology nd Neuroscience, Dlhousie University, Hlifx, NS, Cnd B3H 4R2 *Author for correspondence (sdmo@dl.c) SUMMARY Predtor-induced stress responses re thought to reduce n niml s risk of eing eten. Therefore, these stress responses should enhnce nti-predtor ehviour. We found tht individul insects (the cricket Gryllus texensis) show relile ehviourl responses (i.e. ehviourl types) in plus-shped mze. An individul s ehviour in the plus mze remined consistent for t lest 1/2 of its dult life. However, fter exposure to model predtor, oth mle nd femle crickets showed reduced period of immoility nd n incresed mount of time spent under shelter compred with controls. These chnges could e mimicked y s of the insect stress neurohormone octopmine. These ehviourl chnges proly id crickets in evding predtors. Exposure to model predtor incresed the ility of crickets to escpe live predtor ( erded drgon, Pogon vitticeps). An of octopmine hd the sme effect, showing tht stress hormones cn reduce predtion. Using crickets to study the fitness consequences of predtor-induced stress responses will help integrte ecologicl nd iomedicl concepts of stress. Key words: stress hormone, fight-or-flight, cute stress, niml temperment, niml personlity, Orthopter, Gryllide. Received 26 July 213; Accepted 3 Septemer 213 INTRODUCTION Predtors kill. Animls tht void eing eten will hve n dvntge, provided tht the fitness costs of tht dvntge do not outweigh the fitness enefits. Sensitivity to such fitness costs proly explins the evolution of stress response systems in nimls. By using response tht cn e turned on rpidly nd trnsiently, nimls cn mximize the enefits of nti-predtor dpttions, while minimizing their costs (Orr et l., 21). This perspective suggests tht understnding the costs nd enefits of predtor-induced stress responses will e importnt for interpreting ecologicl nd iomedicl dt on them. Dt from vertertes lrgely support the perspective tht predtor-induced stress responses hve oth costs nd enefits. Acute predtor exposure relily ctivtes stress responses (Apfelch et l., 25). This ctivtion induces coordinted set of physiologicl chnges tht is mostly consistent with the hypothesis tht these chnges prepre the ody for extreme ction (Spolsky et l., 2). Concomitnt ehviourl chnges re thought to increse the ility of nimls to escpe predtors (Apfelch et l., 25). Finlly, studies hve shown tht the use of the stress response entils costs tht re likely to lower fitness; for exmple, the stress response induces reduction in sexul ehviour (Spolsky et l., 2). Nevertheless, it hs proven chllenging to provide detiled ccount of the costs nd enefits of predtor-induced stress responses. For exmple, lthough predtor-induced chnges in ehviour re widely ssumed to help nimls evde predtors, this not een well demonstrted (e.g. Hendrie et l., 1996), nd contrdictory dt exist (e.g. Mher et l., 213). Recent dt suggest tht predtor-induced stress responses re more complicted thn previously elieved. The link etween different components of the verterte stress response system [e.g. sympthetic nervous system nd hypothlmic pituitry drenl (HPA) xis] nd ehviour remins somewht murky (Johnstone et l., 212). In some species, stress responses nd ehviour hve een decoupled (Johnstone et l., 212). Glucocorticoids, trditionlly considered key stress-inducing hormones in vertertes, my e stress-recovery hormones in mny species (Spolsky et l., 2; Johnstone et l., 212; Clinchy et l., 213). For exmple, in tdpoles, cute exposure to predtor decreses corticosterone levels for the first 4 h; corticosterone levels increse ove seline only fter dely of severl hours (Mher et l., 213). Therefore, levels of this stress hormone increse too lte to help tdpoles evde predtor. If corticosterone levels re rtificilly incresed, tdpoles hve reduced survivl ginst predtors (Mher et l., 213). The rpidly ctivting sympthetic nervous system is more likely to e involved in immedite responses to predtors (Wingfield, 23). Unfortuntely, the costs nd enefits of ctivting the sympthetic nervous system hve een little studied (Dickens nd Romero, 29; Breuner et l., 213). This lck of informtion is surprising; much of the complexity regrding the costs nd enefits of stress responses involves long-term effects (Spolsky et l., 2). Although predtor-induced stress responses cn lso hve long-lsting effects (Slos et l., 29; Clinchy et l., 213) tht will complicte clcultion of its cost, it should e possile to demonstrte the mgnitude of the immedite enefit. A comprtive pproch using invertertes my provide importnt complementry informtion out the costs nd enefits of predtor-induced stress responses. Invertertes fce mny of the sme evolutionry pressures s vertertes (e.g. predtion, competition for food nd mtes), ut hve simpler physiologicl systems. They hve roust stress response systems tht in some wys prllel those in vertertes (Roeder, 25; Admo, 28; Admo, 212). For exmple, in insects, the stress response egins with the relese of iogenic mine, octopmine (Orchrd et l., 1993).

2 Predtor-induced stress responses 469 Octopmine is the chemicl cousin of nordrenline (norepinephrine), one of the key cthecholmines used y the sympthetic nervous system (Purves et l., 212). In fct, the octopminergic system ppers to e the functionl equivlent of the verterte sympthetic nervous system (Roeder, 1999; Roeder, 25). The nordrenergic nd octopminergic systems re thought to hve evolved from the sme ncestrl pthwy (Evns nd Mqueir, 25; Cveney et l., 26). Predtor exposure ctivtes the stress response nd increses neurohormonl levels of octopmine (Admo nd Bker, 211). Elevted levels of octopmine produce numer of physiologicl chnges tht enhnce physicl performnce, similr to the effects of verterte stress hormones (Roeder, 1999; Roeder, 25). Octopmine modultes nti-predtor ehviour in eetles (Triolium cstneum) (Nishi et l., 21) nd n or-weving spider (Jones et l., 211). The ehviourl effects of predtor-induced stress cn lso e monitored in insects. As in vertertes (Sih et l., 24; Bell, 27), insects disply ehviourl responses tht re consistent within individuls [e.g. crickets (Hedrick nd Kortet, 212; Niemelä et l., 212)]. These ehviourl styles cn e chnged y predtor exposure (Niemelä et l., 212). Finlly, some of the costs of ctivting the stress response in crickets hve een studied (e.g. Admo nd Prsons, 26). For exmple, n cute stress response results in trnsient decline in disese resistnce (Admo nd Prsons, 26). In this pper, we demonstrte tht individul crickets, Gryllus texensis (Cde nd Otte, 2), show stle nd consistent set of ehviours (i.e. ehviourl strtegy or ehviourl type) (Sih et l., 24; Bell, 27) using modified plus-shped mze (plus mze). We show tht the presence of model predtor shifts cricket s ehviourl strtegy nd tht octopmine is proly involved in mediting this shift. Finlly, we test whether ctivting the stress response or rtificilly rising octopmine levels enhnces escpe from predtors. Such functionl tests re rrely done (Hendrie et l., 1996), ut re importnt for estimting the reltive dvntge supplied y predtor-induced stress responses. MATERIALS AND METHODS Animls Crickets (long-winged G. texensis) were collected ner Austin, Texs, nd hve een mintined s lortory colony for mny genertions with occsionl dditions of fresh nimls from the field. Pellets of dry ct food nd wter were provided d liitum during rering. Crickets were rered t 25±2 C on 12 h/12 h light/drk cycle. Crickets were used etween 1 nd 24 dys fter the moult to dulthood. At this ge crickets re sexully mture (Cde nd Wytt, 1984; Solymr nd Cde, 199) nd within their nturl lifespn in the field (Murry nd Cde, 1995). Adult erded drgons, Pogon vitticeps Ahl 1927, were cptive red nd hd een kept s pets. Berded drgons were kept in 4 gllon (~182 l) terrri. The mient temperture within their enclosure ws kept t 29±2 C during the dy nd 23±2 C t night. A comintion of fluorescent nd incndescent uls provided visile light, UVB nd UVA on 12 h/12 h light/drk cycle. Exo- Terr Plnttion Soil (Hgen Inc., Montrel, QC, Cnd) ws used s sustrte, which ws spot-clened dily. An Exo-Terr Reptile Cve mde from food-grde resin ws provided for shelter. Wter ws provided in lrge wter dish d liitum. Berded drgons were fed crickets (Achet domesticus dusted with clcium powder) every other dy nd given kle, dndelion greens or mustrd greens 6 7 dys week. All studies were pproved y the Animl Cre Committee of Dlhousie University (no. I11-25 for crickets, no. I12-12 for erded drgons) nd re in ccordnce with the Cndin Council on Animl Cre. Chemicls were otined from Sigm Chemicl Co. (St Louis, MO, USA) unless otherwise noted. Evidence for ehviourl type in G. texensis The plus mze ws shped like plus sign nd constructed of lck crylic. It consisted of four rms (L W H cm ech) nd n open centrl re (L W H cm). During the tril, two opposing rms were covered with hevy lck ristle ord, creting drk spces underneth. The other two rms were left uncovered. Crickets prefer covered res (Hedrick nd Dill, 1993). Crickets were gently trnsferred from their home cge to the centrl, uncovered re. They remined under the trnsfer cup for 1 min. The cup ws then removed nd we mesured the time the cricket remined motionless. We clled this time period freezing. Freezing is stereotypic nti-detection response ssocited with predtion voidnce cross tx (Stynoski nd Nole, 212) including crickets (Niemelä et l., 212). We lso mesured the numer of times ech cricket entered n rm of the plus mze s n ssessment of its tendency to explore its environment. We mesured the mount of time ech cricket spent locomoting, oth inside nd outside the covered rms, s proxy for ehviourl ctivity. We lso mesured the time it spent under the covered rms to ssess its tendency to void open spces. Totl tril time ws 1 min. Pilot studies demonstrted tht longer trils (2, 3 or 6 min) did not yield different pttern of results. Crickets (N=12 mles, N=11 femles) were tested four times in the pprtus over 1 dys. The pprtus ws clened with disinfectnt etween trils. At lest 1 dy prior to the plus mze tril, sexully mture crickets (i.e. crickets tht hd mted) (Cde nd Wytt, 1984; Solymr nd Cde, 199) (S.A.A., personl oservtion) were removed from mixed-sex ins nd plced into individul continers (L W H cm) with food nd wter d liitum. Crickets remined in their individul continers for the entire 1 dys. Different people scored the crickets on different tril dys. The dt sheets were filed wy fter ech tril, mening tht ech person running the tril ws unwre of the cricket s previous score. Effects of mock predtor exposure nd octopmine on plus mze ehviour Sexully mture crickets were isolted from the generl colony nd plced into individul continers (1.5 cm dimeter 7 cm) with food nd wter d liitum. Crickets were then ssigned into one of five weight-mtched groups: model predtor exposed, shm exposed, octopmine injected, shm injected nd control. We used the sme procedure s in n erlier study (Admo nd Bker, 211) to mimic predtor exposure. This method relily increses neurohormonl octopmine titres in this species (Admo nd Bker, 211). Briefly, crickets were plced into continer (L W H cm) with loose opque divider seprting it into two hlves. In one hlf of the continer ws the cricket, in the other hlf ws rootic hmster (ZhuZhu Pets, Cepi, St Louis, MO, USA). The rootic hmster mde electronic noises nd moved rndomly out its hlf of the continer, hitting the side s well s the divider. The rootic hmster never contcted the cricket. Arrhythmic virtions, such s those produced y the rootic hmster, induce nti-predtor ehviour in crickets (Dmch, 1989). The crickets tended to run continuously during the tril. -exposed crickets were lso plced into similr continer with divider for 3 min; however, the rootic hmster remined inctive. Control crickets styed in their individul tus nd were not distured efore entering the plus mze. To

3 461 The Journl of Experimentl Biology 216 (24) determine whether chnges in ehviour cused y exposure to the model predtor cn e mimicked y octopmine, fourth group of crickets received n of 2 nmol of octopmine dissolved in 2 μl of wter (see Fields nd Woodring, 1991). -injected crickets were injected with 2 μl of wter. All s were mde with 1 μl Hmilton syringe through the pronotl memrne. Crickets were plced in the plus mze 5 min fter or fter model predtor exposure. Ech cricket ws used in only one tril. Effects of mock predtor exposure on predtor evsion Crickets were isolted, weighed nd ssigned into three groups. The groups were s descried ove: control (mle N=18, femle N=21), model predtor exposed (mle N=19, femle N=2) nd shm-exposed (mle N=18, femle N=21); 5 min fter exposure, crickets were used in the live predtor tril. A second set of trils ws run with control (N=19 mle, N=2 femle), shm injected (N=19 mle, N=2 femle) nd octopmine injected (N=19 mle, N=2 femle) crickets; 5 min fter, crickets were used in the live predtor tril. The predtors were three femle erded drgons, P. vitticeps. The first two drgons were used in n lternting fshion for the first 39 trils, nd the third erded drgon ws used for the finl 39 trils. For ech set of trils, one cricket from ech of the three groups ws plced eneth n overturned opque cup (8 cm dimeter) nd moved to plstic enclosure (L W H cm). The cups were plced in the enclosure long line 11 cm from one of the short wlls (37 cm) nd running prllel to it. A shelter composed of crdord nd duct tpe (L W H cm) rn long this short wll s well. The three cups were equidistnt from ech other nd the long wlls of the enclosure (i.e. cups were out 9 cm prt nd 9 cm from the wll of the continer). The strting position for ech group ws rotted with ech tril. At the strt of the tril, erded drgon ws dded to the enclosure, hlfwy etween the two long wlls of the continer nd 25 cm from the end of the continer wll opposite the crickets. The three cups were then lifted simultneously y mens of rod ttched to ll three cups. The erded drgon ws relesed from its strt position s soon s the crickets were uncovered. The first cricket to enter the shelter nd the first to e consumed y the erded drgon were recorded. Individul crickets could e identified y unique trits such s ntennl length nd cuticulr colour. Trils were videotped. The live predtor trils lsted less thn 1 min. Cptured crickets were killed instntly y the lizrds. For ech tril, only crickets of the sme sex were used. Therefore, no etween-sex comprisons were possile. Sttistics Sttistics were performed using IBM SPSS Sttistics (v19) nd GrphPd Prizm (v5., GrphPd Softwre, L Joll, CA, USA). Behviourl dt were not normlly distriuted, even fter ttempted trnsformtion, with the exception of locomotion. Locomotion ws normlly distriuted fter log trnsformtion. A principl components nlysis (PCA) ws performed on the ehviourl mesures (locomotion, time spent freezing, the numer of rm entries nd the time spent in the covered rms). This method of nlysis is recommended for studies of ehviourl styles (Crter et l., 213). PCA performed for dt reduction cn e used on non-norml dt (Jollife, 22). Only the first component of the PCA hd n eigenvlue ove 1 (score=2.26) nd it explined 56.5% of the totl vrince (N=23 crickets). The vlidity of the PCA ws shown y the mesure of smpling dequcy (Kiser Meyer Olkin=.635) nd highly significnt Brtletts s test of sphericity (P<.1). The resulting first PCA component ws lso non-normlly distriuted, even fter ttempted trnsformtion. Non-prmetric fctoril nlysis (equivlent to two-wy ANOVA) ws performed ccording to Meddis (Meddis, 1984). Non-prmetric tests for homogeneity of vrinces were conducted using the non-prmetric Levene test (Nordstokke et l., 211). The lph criteri were djusted when necessry to ccount for multiple tests on the sme dt set (Benjmini nd Hocherg, 1995) (clculted using BenjminiHocherg.xlsx). RESULTS Evidence for ehviourl type in G. texensis The PCA nlysis demonstrted tht cricket ehviour in the plus mze spnned continuum etween two different strtegies. A high score on the first PCA component descried nimls tht spent little time immoile in the exposed centrl re ut tht spent much of their time locomoting in, nd moving etween, the covered rms. A low fctor score denoted crickets tht remined immoile (in the open) for prt of the tril. These ptterns re evident from the component lodings for ech ehviour: entries,.85; locomotion,.449; time frozen,.87; time within the covered rms,.828. Crickets scoring highly on the first PCA component were deemed to e shelter-seeker/explorers nd low scoring crickets were considered freezers. These scores were consistent nd repetle within individuls (Kendll s coefficient of concordnce W=.8875, P<.1; N=23 crickets, tested four times ech over 1 dys). There were no significnt differences in component scores cross trils (Fig. 1; Friedmn s test=6.18, P=.1), suggesting tht it is stle cross time. This ws tested explicitly with Dunn s multiple comprison post hoc test. There ws no significnt difference in scores etween the first nd the lst (fourth) tril (difference in rnk sum=1., P>.5). This result suggests tht nimls did not hitute to the plus mze. There were no sex differences (Mnn Whitney tests for the shelter-seeker/explorer score, time frozen nd time under the covered rms, P>.5 for ll tests), except tht femles were Shelter-seeking/explorer score Tril 1 Tril 2 Tril 3 Tril 4 Fig. 1. Chnge in shelter-seeking/explorer score over the four trils. There is no significnt trend with time (see Results). The centrl line represents the medin nd the lower nd upper rs represent 1st nd 3rd qurtiles. The error rs denote the rnge.

4 Predtor-induced stress responses Shelter-seeking/explorer score 2 2 Time spent frozen (s) Control Predtor predtor OA Fig. 2. Effect of model predtor exposure or octopmine on shelter-seeking/explorer score. Both mock predtor exposure nd octopmine (OA) increse shelter-seeking/explorer score. The centrl line represents the medin nd the lower nd upper rs represent 1st nd 3rd qurtiles. The error rs denote the rnge. Brs with different letters ove them re significntly different from one nother. c Control Predtor predtor c OA Fig. 3. Time spent frozen fter exposure to mock predtor or octopmine (OA). The centrl line represents the medin nd the lower nd upper rs represent 1st nd 3rd qurtiles. The error rs denote the rnge. Brs with different letters ove them re significntly different from one nother. somewht more ctive thn mles (log locomotion dt, F 1,21 =6.34, P=.2); however, this result is only mrginlly significnt fter correction for multiple tests [fter Bonferroni correction, lph criterion (.5 level)=.125 (Benjmini nd Hocherg, 1995), no significnt results]. There ws no correltion etween the shelterseeker/explorer score (men over four trils) nd weight (Spermn s r=.13, P=.64, N=23). Effects of predtor exposure nd octopmine on plus mze ehviour A PCA ws performed seprtely on these dt, extrcting single component with n eigenvlue of 2. explining 49% of the vrince. This component showed the sme reltionship to the four ehviourl vriles s descried in the section ove. It correlted positively with strtegy of multiple entries, greter locomotion nd time spent under the covered rms, ut correlted negtively with time spent frozen in the centrl re (component lodings: entries,.682; locomotion,.622; time frozen,.747; time in covered rms,.744). Across the five tretment groups [model predtor exposed (mle N=17, femle N=17), shm exposed (mle N=17, femle N=17), octopmine injected (mle N=16, femle N=17), shm injected (mle N=16, femle N=16) nd control (mle N=17, femle N=17)], there were no significnt differences due to sex for ny of the ehviourl mesures (Mnn Whitney tests, ll P>.3). Therefore, mle nd femle dt were pooled for ech tretment group nd nlysed using Kruskl Wllis test followed y Dunn s multiple comprison post hoc test where pproprite. There were significnt differences in shelter-seeking/explorer scores cross the five groups (Fig. 2, Kruskl Wllis, KW=4.7, P<.1). Dunn s multiple comprisons showed tht oth predtor-exposed nd octopmineinjected crickets hd higher scores thn did controls (P<.1). To exmine the chnges in more detil, we studied the individul ehviours, except for numer of rm entries ecuse tht vlue tended to e low in ll groups. Time spent frozen ws significntly different cross groups (Fig. 3, Kruskl Wllis, KW=39.2, P<.1). Model predtor-exposed nd octopmine-injected crickets oth spent less time frozen thn did control, shm-injected or shm-exposed crickets (Dunn s rnk sum multiple comprison test, P<.5). -injected nd shm-exposed crickets were frozen for significntly shorter time thn controls (Dunn s rnk sum multiple comprison test, P<.5). The medin time spent within the covered rms ws lso significntly different mong groups (Fig. 4, Kruskl Wllis, KW=53.8, P<.1). The time spent within the covered rms ws not significntly different etween the model predtor-exposed nd octopmine-injected crickets. Model predtor-exposed nd octopmine-injected crickets oth spent more time within the covered rms thn did shm-injected, shm-exposed or control crickets (Fig. 4, Dunn s rnk sum multiple comprison test, P<.1). The medin time spent locomoting ws not significntly different cross groups (Kruskl Wllis, KW=5.63, P=.229). Vriility of the shelter-seeker/explorer score ws less in the predtor-exposed or octopmine-injected groups thn in controls (non-prmetric Levene s test, F 4,162 =4.7, P=.1; Fig. 2). Effects of predtor exposure on predtor evsion Model predtor-exposed crickets (N=22/39) were significntly more likely to e the first individuls to rech shelter first thn were either the control (N=8/39) or shm-exposed (N=9/39) groups (chi-squred test, χ 2 2=14.8, P=.5). Similrly, octopmine-injected crickets (N=19/39) were significntly more likely to e the first individuls to rech shelter first thn were either the control (N=7/39) or shminjected (N=13/39) groups (chi-squred test, χ 2 2=8.3, P=.2). Model predtor-exposed crickets (N=19/39) were significntly more likely to survive exposure to predtor thn either the control

5 4612 The Journl of Experimentl Biology 216 (24) 6 c 6 Time spent under covered rms (s) 4 2 % Survive 4 2 Control predtor Predtor OA Control Predtor predtor (N=9/39) or shm-exposed (N=11/39) crickets (Fig. 5, chi-squred test, χ 2 2=6.4, P=.5). Similrly, octopmine-injected crickets (N=19/39) were significntly more likely to survive exposure to the predtor thn either the control (N=8/39) or shm-injected (N=12/39) crickets (Fig. 5, chi-squred test, χ 2 2=7.4, P=.25). DISCUSSION Individul G. texensis crickets exhiited distinct ehviourl types in the plus mze tht were consistent for t lest hlf of their dult lifespn. However, these strtegies were not immutle; they were sensitive to the presence of predtor (Fig. 2). Exposure to mock predtor shifted crickets wy from using freezing s n nti-predtor strtegy nd incresed the time they spent under cover (Figs 2 4). Rodents show similr pttern of ehviour in the elevted plus mze (Apfelch et l., 25; Eilm et l., 212; Hcquemnd et l., 213). Predtor exposure lso reduces freezing in relted species of cricket tested using different protocol (Niemelä et l., 212). These results re consistent with current thinking out defence strtegies. Defensive ehviours re thought to exist within hierrchy (Hnlon nd Messenger, 1996). Freezing occurs during low thret events, ut if predtor continues to dvnce, nimls flee (Hnlon nd Messenger, 1996). Cricket defensive ehviour fits this frmework; crickets freeze when the predtion risk increses from low to moderte, proly to void detection y nery predtors. However, if n ttck is imminent, crickets flee. Our study, nd those of others (e.g. Apfelch et l., 25; Niemelä et l., 212), shows tht the thret level needed to shift nimls from freezing to fleeing cn e lowered y previous exposure to predtor. This shift is proly dptive ecuse eing ttcked y predtor in the immedite pst is likely to e good predictor tht nother ttck is imminent if predtor cues re-occur. Being le to lter ntipredtor ehviour depending on the environment is likely to provide importnt fitness enefits (Storm nd Lim, 21). Our results lso provide support for the concept tht the stress response is continuum (e.g. Hcquemnd et l., 213), s might OA Fig. 4. Time spent under the covered rms fter exposure to mock predtor or octopmine (OA). The centrl line represents the medin nd the lower nd upper rs represent 1st nd 3rd qurtiles. The error rs denote the rnge. Brs with different letters ove them re significntly different from one nother. Fig. 5. Percentge of crickets tht survive the encounter with the predtor. Smple size is N=39/group except for the control r. This r represents the verge of the two control groups (from the first nd second trils, N=78). Mock predtor-exposed nd octopmine (OA)-injected crickets were significntly less likely to e eten thn controls. e expected if it is involved in determining the pproprite level of defensive ehviour. Our shm controls sometimes hd ehviourl scores tht were intermedite etween the unhndled controls nd the predtor-exposed or octopmine-injected crickets (e.g. Fig. 2). Similrly, hndling stress in irds induces smller stress response thn predtor ttck (Pkkl et l., 213). These results suggest tht the stress response is not n ll-or-none event, ut tht it cn hve grded effects depending on the severity of the thret. After mock predtor exposure or octopmine, not only did the crickets nti-predtor ehviourl strtegy shift ut lso vriility in ehviourl style in the plus mze declined compred with controls (e.g. Fig. 2). These results suggest tht under norml conditions, numer of ehviourl strtegies my e dptive, ut in the presence of predtors, optiml solutions quickly converge on much smller suset of strtegies. Such shifts in nti-predtor ehviour cn e long lsting nd, in crickets, cn e trnsmitted trnsgenertionlly (Storm nd Lim, 21). Injection of octopmine led to reduction in the use of immoility (i.e. freezing) s n nti-predtor ehviour (Fig. 2). Similrly, rising octopmine levels decresed the use of immoility s n nti-predtor ehviour in oth n or-weving spider (Jones et l., 211) nd the eetle T. cstneum (Nishi et l., 21). These results suggest tht octopmine my e involved in ltering the threshold for different defensive ehviours in mny rthropods. However, the lck of effect of octopmine on locomotion seems puzzling, given its postulted role in mediting generl rousl (Roeder, 1999). Nevertheless, octopmine lso hd no ffect on generl locomotion in the or-weving spider (Jones et l., 211). We hypothesize tht one of the effects of elevted octopmine is tht it shifts nti-predtor ehviour towrds defensive ehviours used for more serious threts. In our plus mze study, crickets injected with octopmine my e more inclined to flee into hiding thn controls, ut once in drk, sfe plce, they will e less likely to move out of it. Therefore, in our plus mze, with its covered drk res, octopmine-injected crickets my e less likely to express elevted locomotion. Model predtor-induced chnges in ehviour incresed the ility of crickets to evde n ctul predtor (Fig. 5). Exposure to model predtors is known to ctivte the stress response in this species (Admo nd Bker, 211). Moreover, n of the

6 Predtor-induced stress responses 4613 stress neurohormone octopmine lso enhnced nti-predtor ehviour (Fig. 5). These results suggest tht predtor-induced stress response ssists crickets in evding predtor. Therefore, one importnt enefit of stress hormones in insects ppers to e to promote survivl during predtor ttck. This pper supplies rre exmple of the mgnitude of the survivl enefit due to predtor-induced stress response. Previous work hs demonstrted some of the costs of ctivting the stress response in crickets. For exmple, flight-or-fight ehviours induce lrge increse in metolic rte (e.g. Hck, 1997), leding to the consumption of scrce resources. Octopmine promotes the consumption of these resources y inducing the relese of lipid from ft stores (Fields nd Woodring, 1991). This moiliztion requires energetic nd moleculr resources (Ntion, 28). Such metolic costs re not trivil for smll nimls. For femles, the mount of lipid moilized during stress response (Admo et l., 28) is equivlent to 1.4% of the resources in n egg (see Shoemker nd Admo, 27). When resources re scrce, dditionl demnds decrese reproduction in this species (Admo nd Lovett, 211). Acute ctivtion of the stress response lso induces the reconfigurtion of physiologicl networks tht results in reduction in function for numer of physiologicl systems (e.g. immunity) (Admo et l., 28; Admo, 29; Admo, 21). The full costs of these shifts remin unknown. This pper shows tht crickets re useful models for studying the costs nd enefits of predtor-induced stress responses. Although studies in vertertes (e.g. Spolsky et l., 2; Wingfield, 23; Øverli et l., 27; Romero et l., 29; Oswld et l., 212; Romero, 212) continue to increse our understnding of these stress responses, complementry exmintion of them in invertertes cn dd n importnt perspective. Crickets, with their short genertion time, lck of prentl cre nd strightforwrd stress response systems re trctle models for ddressing questions regrding the fitness consequences of predtor-induced stress responses. Such informtion will e importnt in integrting ecologicl nd iomedicl concepts of stress. ACKNOWLEDGEMENTS We thnk M. Acker nd A. McKeen for mintennce of the crickets nd M. Acker, M. D Angelo, J. Bker, G. Logn, M. E. O Lery, D. Rossetti nd R. Wheeler for ssistnce running the ehviourl trils. AUTHOR CONTRIBUTIONS B.M. helped design the live predtor trils, rn some of the ehviourl trils nd criticlly red the mnuscript. I.K. helped design the plus mze trils, rn some of the ehviourl trils nd criticlly red the mnuscript. S.A.A. proposed the originl concept, designed the overll tril, nlyzed the dt, wrote the pper nd helped run some of the ehviourl trils. COMPETING INTERESTS No competing interests declred. 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