CEC Research | https://doi.org/10.21973/N3MD4J Fall 2017 1/6

Defensivedisplaysandchemicalsecretionsofdarkling

beetlesintheMojaveDesert

TaniaAguilar1,DidraFelix2,MeghanProctor3,AthenaSchlereth4

1UniversityofCalifornia,Davis,2UniversityofCalifornia,SanDiego,

3UniversityofCalifornia,SantaCruz,4UniversityofCalifornia,Berkeley

ABSTRACT

Manyorganismsoften relyonchemicaldefenses todeterpredators.However,the way in which invertebrates utilize chemical defenses has been relativelyunderstudied. Darkling beetle species of the Eleodes and Coelocnemis generaexhibitdefensiveposturingfollowedbymalodorouschemicalsecretionstosignaltheir unpalatability towards potential predators.However, themetabolic costsandtrade-offsassociatedwiththesemechanismsareunknown.Itisalsounclearwhether certain beetle species utilize chemical defenses more readily thanothers. We examined the defensive displays and secretions of four darklingbeetlesspecies.Wealsoexaminedtheeffectofcaptivityondefensivebehaviorby comparing responses in beetles challenged in situ versus beetles thatwereheldcaptive.Wefoundthatbeetleschallenged incaptivityexhibiteddefensiveposturing for a significantly shorter time than beetles challenged in situ. Inaddition,onespecies,Eleodesarmatus,requiredasignificantlylongerperiodofagitationbeforeodorreleasecomparedtotheother threespecies.Ourresultssuggestthatpriorstresscancausethebeetlestochooseevasionoverdefensiveposturingandthatdifferentspeciesutilizedifferentstrategiesintheirdefensivesecretions.

INTRODUCTION

Chemical defense mechanisms haveevolvedinanumberoforganismsasaneffectivemethod in reducing predationrisk (Šanda et. al. 2012). Chemicaldefenses in organisms are eithersynthesized endogenously orsequestered exogenously from the

environment and can broadly range intheir level of intensity, from beingdistastefulandunpalatable(e.g.stingingnettle, striped skunk, etc.) to highlytoxic and even lethal to predators (e.g.tetrodotoxin in pufferfish, roughskinned newts, blue-ringed octopus,among others). Chemical defenses are

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often accompanied by warningbehaviors that signal the possession ofchemical weaponry to predators(Skelhorn and Ruxton 2007). Thesebehavioral adaptations often come atan ecological cost, such as reducedabilitytoretreatfrompredatorsquickly,larger body sizes and diurnal activity.However,chemicaldefensemechanismsandwarningbehaviorscanbecriticalforsurvivorship despite these costs (Hatleand Townsend, 1996). The use ofdefensive secretions is an effective butcostly weapon for Eleodes andCoelocnemis beetles, and thus, theseverity of their defensive behavior(warning display versus chemicalrelease) may vary with perceivedpredation risk. Specifically, releasingdefensivesecretionsareassociatedwithmetabolic costs of synthesizing thechemical. While beetles can secretesufficient discharge from defensiveglandsmorethanonceduringanattack,they typically only discharge oncebecause residual secretions thatpersiston the abdomen can sufficiently deterpredators and depleted glands mayrequire several days to replenishcompletely (Eisner and Meinwald,1966).Therefore,controllinghowmuchdefensive secretion is released versusthe less costly behavior of exhibiting adefensive posture without chemicalsecretion during an attack is animportantadaptationforthesebeetles.Members of the genus Eleodes and

Coelocnemis, also known as darkling orpinacate beetles, possess repugnatoryglands that produce malodorousdefensive secretions that allowpredators to quickly learn todiscriminate against these beetles

(Eisner and Meinwald, 1966). Ifthreatened by a predator, Eleodes andCoelocnemis beetles will dischargechemical defenses from therepugnatory gland as a spray. Thisdisplay,however,maymake thebeetlemore vulnerable as it requires them tofacetheirheadintotheground.Assuch,thesebeetlespossessanotherchemicaladaptation as a final deterrent againstpredation—the blood of these beetlesalso consists of repellent substancesthat make them unpalatable topredators (EisnerandMeinwald,1966).Thus, these repellent substancesincrease their chances of surviving apredatoryattack.While we know that these beetles

utilize chemicaldefense, there is a lackofunderstandingregardinginterspecificdifferences in defense displays amongbeetle species. These unknowndifferences between interspecificdifferencesincludedefensiveresponses,differences in chemical secretion, andbody structures. In addition, there isalso a lack of understanding regardingthe effects of captivity on organismsthat utilize chemical defenses.Individuals can vary widely in theirresponses to captivity. They are oftenhighly susceptible to stress, haveshorter lifespans and may havereproductive problems, reducing theiroverall survivorship (Mason, G. 2010).Thislackofunderstandinghasledustoinvestigate these knowledge gaps bylooking at the beetles of GraniteMountaintoseeifchangesinstressanddifferences among species affectdefensivesecretionresponsetime.We investigated various trade-offs in

chemical defense behaviors in beetles

CEC Research | https://doi.org/10.21973/N3MD4J Fall 2017 3/6

(representatives from Eleodes andCoelocnemis). We first asked whetherthedurationofdefensiveposturingandtime until chemical release would varyamongbeetlespecies,potentiallyduetointerspecific differences in size.Secondly, we asked how captivityaffects defensive chemical secretion.We hypothesized that wild beetleswouldbemorepronetopredationandwouldthereforeexhibitlongerdefensivepostures. We also hypothesized ashorter time until defensive chemicalsecretion in captivebeetles versuswilddue to the physiological stress. Insummation,allof thesewouldallowusto better infer how stress andinterspecificdifferencesamongstbeetlespeciesaffectresponsetimetostimulusand theway inwhich beetles prioritizetheseecologicaltrade-offs.

METHODS

StudySystem

We conducted our study fromNovember 3–8, 2017, at SweeneyGranite Mountains Desert ResearchCenter (34.7813° N, 115.6538° W),located in the Mojave Desert, KernCounty, California. Our study areaconsisted of desert scrub habitatdominated by cholla (Cylindropuntiaacanthocarpa), cat-claw acacia(Senegaliagregii),creosotebush(Larreatridentata), and Mojave yucca (Yuccashidigera).

ExperimentalDesign

In order to examine differences inchemical defense behavior amongbeetlespecies,weconductedchallengeexperiments with beetles encountered

in situ. We selectively sampled beetlesbywalkingalongroadsandtrailsduringearlymorningsandlateafternoonwhenbeetles weremost active (Smith et. al.2014).Foreachencounteredbeetle,wefirstrecordedhowclosewecouldgettothe beetle before it displayed adefensive posture. If there was noreactiontoourpresence,weconductedconsecutive challenges of perturbationon the beetle, with each challengesimulating an increasing level ofpredation risk. During the firstchallenge, we aggressively tapped thebeetle on top of their abdomen usingmetalforcepsandtimedthedurationoftheirdefensivedisplay.Whenthebeetlelowered its abdomen and began toretreat, we conducted a secondchallenge by tapping the beetle twicemore with our forceps and againrecorded the duration of their seconddefensive display. After the beetlelowered its abdomen again, weconducted a final challenge bycontinuallytappingitonitsabdomenina consistent rhythm and recorded theduration of time until it released itsdefensive secretions or exposed itsrepugnatory gland (without directchemical secretion). Following thesechallenges,wecollectedthebeetleandstored it in a 4oz. specimen cup tobeused in further challenge experiments,asdescribedbelow.Wefurtherinvestigatedtheeffectsof

captivity on behavioral defensiveresponse in beetles collected from insitu challenges, as well as beetles thatwere opportunistically collected in thereserve. All beetles were kept incaptivityforonedaybeforeundergoingfurther challenges in order to ensure

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that all captive beetles underwent thesame stress induced environment. Weconducted challenges with thesebeetles according to the sameprotocoldescribedabove.Forbothcaptiveandwildbeetles,we

calculated total time of interaction asthe sum of times from all threetreatments and used this to measureinterspecific differences in defensivebehavior.Weusedelectroniccaliperstomeasurephysicalcharacteristicsofeachbeetle representative of interspecificdifferencesinsizeusingthoraxwidthasaproxybecauseitwasthemostreliableform of morphological measurement.We also recorded the number ofantennasegments,legspikes,andtarsalclaws of each individual to assist inspeciesidentification.

StatisticalAnalysis.

Weused JMP Pro 13 software for alldata analyses. For these analyses, weperformed ANOVA tests comparingmorphological measurements amongbeetle species to determine if therewere interspecific behavioraldifferences. We conducted a t-test toexamine interspecific differences induration of defensive display bycomparing total time until chemicalrelease in E. armatus versus all otherbeetle species in our study due to itsprevalence in our study site. Weconducted a t-test to investigate theeffect of captivity on the duration ofdefensive display for the first andsecondchallenge.Weusedaone-tailedt-test for these analyses because ourapriori hypothesis predicted thatcaptivitywoulddecreasethedurationofadefensivedisplay.

RESULTS

Outof the38beetles sampled in thisstudy, 18 were Eleodes armatus, 16wereE.dentipes,twowereE.obscurus,and two belonged to genusCoelocnemis. We found that eachspecies of beetle had a unique meanthoraxwidth(N=38,P<0.0001;Figure1). We found that E. armatus took alonger time to secrete their defensivechemicals compared to theother threespecies (N = 38, P = 0.02; Figure 2).Additionally, we found that beetlesencountered in situ held a longerdurationofdisplaycomparedtobeetleschallenged in captivity during both theinitialchallenge(N=38,P=0.04;Figure3) as well the subsequent secondchallenge(N=38,P=0.03;Figure4).

DISCUSSION

Our results support our researchquestion that there are differences indefensive behavior in wild and captivebeetles.Weobserved thatwild beetleswill display defensive postures longerthan those in captivity, with captivebeetles opting to flee. Whenreintroduced to stimulus, wild beetleswould still be in a defensive displaylonger than the captivebeetle.Overall,we saw a trend that captive beetlestooklesstimetoreleasetheirdefensivedischarge compared to wild beetles.Thismay be due to increased stress incaptivity (Mason, G. 2010).Furthermore, defensive chemicalsecretion differed among differentspecies,withE.armatustakinglongerto

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Figure1.Meanthoraxplatewidth±seofbeetlespecies.Barsrepresentmeans+1S.E.M.

Figure2.Meandurationoffirstandsecondthreatdisplay±seofbeetlewildandcaptive.

Figure3.MeanodorreleasetimeofE.armatusversusallotherspeciessampled.Errorbarsrepresent±1standarderror.

secrete their chemical defense. Thismay suggest E. armatus is betteradapted to theenvironmentbecause isoneofthelargestbeetlesandthemostprominentinthearea.In addition, we were able to identify

the presence of beetles (Eleodesdentipes, Eleodes obscurus, andCoelocnemis) that had not beenrecorded at Granite by comparingphysical characteristics such as meanthorax width. It is unclear how manyspecies of Eleodes and Coelocnemisoccur in the Mojave Desert, but wewereabletoconfirmthatthereismorethan just theE.armatus species,whichis currently theonlyEleodesspeciesonthe Sweeney Granite Mountain DesertResearch Center invertebrate specieslist. We suggest that more researchshould be done to distinguish thedifferences and similarities in thegeographicdistribution,physiology,andbehavior between species of EleodesandCoelocnemis.Future research can focus on the

composition of repugnatorial secretionbetween species, especially those thatexhibit Müllerian mimicry. WhileMüllerian mimics have retained thesedefensive glands through convergentevolution, it would be interesting toexplore the similarities and differencesbetweeneachspeciesandthechemicalcompositionofthecompoundsfoundintherepugnatoryglandthatmakestheseorganisms unpalatable (Eisner andMeinwald, 1966). Further researchcould also focus on the phylogeneticrelationships among these beetlespecies. Future studies could alsoexplore the similarities of defensivechemical composition in plants as well

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ascompoundsfoundinbeetledefensivesecretion in areas of overlappingdistribution.Finally,furtherresearchonthe implications of captivity onorganisms, specifically those withdefensive chemical secretions, isneeded to better inform catch-and-release studies and improve individualwelfareoftheorganism.The costs and benefits of these

beetles’ defensive posturing andchemical secretion has not beenquantified in prior research. Theirchemicaldefensesareacostlyresource;we would expect the beetles to haveadaptedbyonlyutilizingitwhentheriskof actual predation is high enough.There is a choice of “fight or flight” insuch situations. We observed thatbeetles, which experienced the priorstressof captivity, chose to fleeoverallrather than display their defensiveposture. This result suggests thatbeetles, which were attacked,previously may choose to evade thenextpredatorratherthandefend itself.This highlights the costliness of theirchemical defenses and sheds insightintowhich situationsbeetles choose toevadeordefendthemselves.

ACKNOWLEDGEMENTS

This work was performed at theUniversity of California’s SweeneyGranite Mountains Desert ResearchCenter,doi:10.21973/N3S942.

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