84 Nematology, Florida, Gainesville, FL32611,USA([email protected]) University of Entomologyand of *Author at present address:Department for correspondence Organismal Biology, OhioState University, Columbus, OH43210,USA. Evolution, Ecology and of EntomologyandDepartment of Department maritime regions(Convey, 1996a;Convey, 2013). established inAntarctica,and mostofthesearerestrictedto life formonEarth,onlyahandfulofspecieshavesuccessfully (Kennedy, 1993).Whereasarthropodsarethepredominantterrestrial the biggestchallengeconfrontingterrestrialarthropodsinAntarctica unavailable formuchoftheyear, thuswater availabilityisperhaps and Lee,1981).Furthermore,waterisfrozenbiologically subzero temperaturescanbeexperiencedanytimeofyear(Baust sea limitstemperatureextremes,winterlowsexceed–40°C and of .EveninmaritimeAntarctica,whereproximity to the the environmentalonslaughtsmorechallengingthancontinent encounter someformofenvironmentalstress,perhapsnowhere are distribution ofterrestrialarthropods.Whileinsectsonallcontinents is oneoftheprimaryconstraintsgoverningabundance and Environmental stress,intheformofbothbioticandabiotic Environmental stress,Physiology KEY WORDS:Antarctica,Coldtolerance,Dehydration, of freezing.Incontrast,thetwobest-studiedAntarcticinsects, dehydration toextendtheirsupercoolingcapacityandreducetherisk some ofthesemicroarthropodsarecapablecryoprotective deep supercooling,insomecasessupercoolingbelow–30°C.Also, temperatures, mitesandCollembolaarefreeze-intolerantrelyon physiology ofterrestrialarthropodsinAntarctica.To survivelow review thecurrentstateofknowledgeregardingenvironmental to copewithextremesintemperatureandwateravailability. Here,we Antarctica. Antarcticarthropodshaveevolvedasuiteofadaptations success ofarthropods,andnowhereisthismoreapparentthan Abiotic stressisoneoftheprimaryconstraintslimitingrangeand Nicholas M.Teets* andDavidL.Denlinger terrestrial Antarcticarthropods Surviving inafrozendesert:environmentalstressphysiologyof REVIEW © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,84-93doi:10.1242/jeb.089490 of coldandwaterstressintheAntarctic. metabolic restructuringandcellrecyclingpathwaysaskeymediators cuticular andcytoskeletalrearrangements,heatshockproteins, Some commonthemesthatareemergingincludetheimportanceof on theunderlyingmechanismsthatgovernextremestresstolerance. their infancy, butseveralrecentstudiesarebeginningtoshedlight Molecular studiesofAntarcticarthropodstressphysiologyarestillin cuticles buttolerateupwardsof50–70%lossbodywater. loss acrosstheircuticle,whileamajorityhavehighlypermeable some accomplishthisbycuticularmechanismstominimizewater among Antarcticarthropodsisextremetoleranceofdehydration; hardening tocopewithdecreasesintemperature.A commontheme tolerant year-roundandrelyonbothseasonalrapidcold- Introduction ABSTRACT antarctica and Eretmoptera murphyi,arefreeze- Peninsula inanarealessthan0.1 Friesea grisea)wasfoundunderasinglerockontheAntarctic 2 millioncollembolaneggs(from local abundancecanbeveryhigh.Forexample,amassof over (Convey, 1992).DespitethelackofspeciesdiversityinAntarctica, in maritimeAntarcticafromsubantarcticSouthGeorgia inthe1960s Allegrucci etal.,2012)],wasaccidentallyintroducedtoSignyIsland considered bysometobeamemberofthegenus to Antarctica.A thirddipteranspecies, South America, (Convey andBlock,1996).AsP. stenenii species, themidges ; trueinsectsnativetoAntarcticaconsistoftwochironomid most glaringaspectofAntarctica’s terrestrial faunaisitslackoftrue which areendemictothecontinent(Greenslade,1995).Perhaps Antarctica arehometo15speciesofCollembola,againmost maximum (Mortimeretal.,2011). Maritimeandcontinental endemic andseemtohaveestablishedpriorthelastglacial half areparasiticticksandmites.Nearlyallfree-livingspecies by over100species(MarshallandPugh,1996),ofwhichroughly 2013). Acariisthemostdiversetaxononcontinent,represented dominated bymitesandcollembolans(Convey, 1996a;Convey, The predominantarthropodsinAntarcticaaresoilmicroarthropods, distinguish Antarcticarthropodsfromtheirtemperatecounterparts. to continuesearchingfortheuniquemolecularcharacteristicsthat provide afewsuggestionsforfuturedirections,inparticulartheneed launched Antarcticresearchintoanewera.Inthefinalsection,we molecular biology, particularly the‘omics’ revolution,have havebeenlimitedinscope.However, recentadvancesin mention difficult toaccess)species,molecularstudiesofAntarctic stress responseinAntarcticarthropods.Asnon-model(notto we focusonthebiochemicalandmolecularunderpinningsof environmental stresstoleranceinAntarcticarthropods.Inparticular, . only bediscussedwhenthere isclearoverlapwitharthropod microfaunaarebeyond thescopeofthisreviewandwill et al.,2011). However, thephysiologicaladaptationsofthesenon- diversity ofthisgroupmaybesubstantially underestimated(Nielsen endemic toAntarctica(Convey etal.,2008);thetruespecies maritime andcontinentalAntarctica, allofwhichappeartobe Additionally, anumberofnematodespeciesarefoundinboth inhospitable toarthropods(ConveyandMcInnes,2005). free areasthroughoutAntarctica,evenincontinentalregions arthropods (Telford etal.,2008), arefoundinhighabundanceice- example, tardigrades,membersofacloselyrelatedphylumbasal to species diversity, seemtoflourishinharshinlandenvironments.For but therearesometaxaofmicrofaunathat,despitelowoverall Overview of Antarctic arthropods Antarctic of Overview In thisreview, wesummarize thelimitsandmechanismsof Arthropods aregenerallyconcentratedinmaritimeenvironments, B. antarctica is consideredtheonlyinsectendemic

m 2 Cryptopygus antarcticus (Schulte etal.,2008). Eretmoptera murphyi and is alsofoundinsouthern Parochlus steinenii Belgica [now (see and

The Journal of Experimental Biology ( collembolans), ofthethreechironomidspeciesoncontinent,two representative speciesarefromapteroustaxa(i.e.mitesand noteworthy featuresofAntarcticarthropods.Whilemost exhibit diapause,evenintemperateregions. has onlybeenexaminedinnon-insecttaxa,whichtypicallydonot unpredictable growingseasons.However, thecapacityfordiapause allows Antarcticspeciestotakeadvantageoftheextremelyshort, on quiescencetoendureunfavorableperiods(Convey, 1996b).This cycles, Antarcticarthropodstypicallylackdiapauseandinsteadrely chironomids (ConveyandBlock,1996).Inconjunctionwithlonglife antarctica multiple generationsperyear. Similarly, theAntarcticmidge (Burn, 1981),whereastemperatecollembolanstypicallycomplete the Antarcticmite energy fromgrowthandreproduction.Forexample,populationsof (see belowforexamples)areenergetically costly, thusdiverting towards environmentalstressprotection.Manymechanisms of Antarcticarthropodlifehistoriesisthelarge investmentofenergy sporadic, inconsistentnutrientsources.A finalnoteworthyattribute generalist herbivoresanddetritivores(Davis,1981),thrivingon resources (Hoggetal.,2006).A majority ofAntarcticarthropodsare there isthoughttobelittleinter- andintra-specificcompetitionfor stressors areconsideredtheprimaryselectivepressureinAntarctica, reducing thesurfaceareatovolumeratio.Becauseenvironmental dispersal intounfavorableenvironmentsandtoconserveheatby isolated, windsweptregions,andservestopreventaccidental (Convey andBlock,1996).Brachypteryisacommonadaptationin quantified. species, thefitnesscostsofelevatedstresstolerancehavenot been tolerance (Convey, 1998).However, foramajorityofAntarctic differential resourceallocationtowardsenvironmentalstress than populationsinmaritimeAntarctica,presumablybecauseof climates areabletodivertsignificantlymoreenergy toreproduction Antarctic environments, thecoldtoleranceof Antarctic arthropods and Diptera). predominant arthropodtaxaon the continent(i.e.Acari,Collembola specific examplesandgeneral principlesfromeachofthe Rather thancomprehensivelyreviewing everystudy, wewill provide strategies andlimitsofstress toleranceinAntarcticarthropods. extreme stresstolerance.Inthissection,wereviewthe basic recent studieshavebeguntounravelthemolecularmechanisms of tolerance havebeenextensivelystudiedinAntarcticarthropods, and established onthecontinent.Thus,basicmechanismsof stress stress tolerance,becausesofewspecieshavebecomesuccessfully arthropods areexcellentmodelsfortheevolutionaryphysiology of physiological costforAntarcticarthropods.Also, As mentionedabove,mechanismsofstresstoleranceareamajor antarcticus (Convey, 1996a).Forexample, theAntarcticcollembolan Antarctic arthropodstakemultipleyearstocompletetheirlifecycle Antarctica leadtoextremelyshortgrowingseasons,soinmanycases of Antarcticarthropodsislifespanextension.Thelowtemperatures mentioning here.Perhapsthemostconspicuouslifehistoryadaptation reviewed extensively(Convey, 1996a;Convey, 2010),theyareworth While lifehistoryadaptationsofAntarcticinvertebrateshavebeen REVIEW Environmental stress tolerance of Antarctic arthropods Antarctic of tolerance Environmental stress arthropods Antarctic Basic ecophysiologicalcharacteristicsof B. antarctica Aside fromprolonged,flexiblelifestyles,thereareacoupleother With lowtemperature beingthemostconspicuousfeatureof has a2-yearlifecycle,whichisrareamongthe takes anestimated3–7 and Alaskozetes antarcticus E. murphyi)aresecondarilybrachypterous

years tocompleteitslifecycle from mildsubantarctic C. B. approximately of bothB.antarctica the onlyknownfreeze-tolerant arthropodsonthecontinent.Larvae and introduced In contrast,theothertwoAntarctic insects,endemic temperature, perhapsexplaining itsmodestlevelofcoldtolerance. ponds andlakes,P. steinenii aquatic midgethatoverwintersaslarvaeatthebottomoffreshwater hardiness ofP. steinenii other Antarcticdipterans(seebelow).However, thewintercold Sjursen, 2001).Theseabirdtick as wintermicrohabitattemperaturescanreach not beenmeasured,theyareassumedtobemuchlowerthan and Sinclair, 2002).Whileoverwinteringsupercoolingpointshave over thecourseofsummer, likelybecauseoffeeding(Sjursen supercooling pointaround and Block,1980).Anothermite, glycerol andremovalofice-nucleatingparticlesfromthegut(Young around Antarctic mite on supercoolingtosurvivesubzerotemperatures.Forexample,the mites (OrderAcari),areallfreeze-avoiding(Sømme,1981)andrely has beenextensivelystudied.Themostprimitivegroup,ticksand (and lethal temperaturesof and capacity, withsummersupercoolingpointsaround is freeze-susceptibleandhasrelativelymodestsupercooling mechanisms ofoverwinteringcoldtolerance.Themidge temperature (Fig. involves cellularprotectionagainstthedamagingeffects oflow to as‘RCH’,itappearsbedistinctfromRCH day (Sinclairetal.,2003).Whilethisphenomenonhasbeenreferred nighttime supercoolingpointsare>10°Clowerthanthoseduringthe clearance; forexample,intwospeciesofAntarcticCollembola, can beobservedasaresultofdailyrhythmsfeedingandgut et al.,2000).Inthefield,diurnalvariationsinsupercoolingpoints supercooling pointareachievedprimarilybygutclearance(Worland Gut clearancealsoplaysaroleduringRCH,asrapiddecreasesin depressing theirsupercoolingpoint(Worland andConvey, 2001). collembolans arecapableofrapidlyincreasingcoldtoleranceby points (Worland andConvey, 2008).Likemites,Antarctic molt periodthatresultsingutclearanceanddecreasedsupercooling Decreasing temperaturestriggerprogressionintoanon-feeding,pre- collembolans appearstobeprimarilyregulatedbythemoltcycle. et al.,2006).SeasonalregulationofsupercoolingpointsinAntarctic Sjursen, 2001)andamoderatedegreeofthermalhysteresis(Sinclair combination ofextremelyhighhemolymphosmolality(Sinclairand Block, 2003).Extensivesupercoolinginthisgroupisachievedbya (Cannon andBlock,1988;Worland andConvey, 2001;Worland and capacities, withsupercoolingpointsaround avoiding. Generally, collembolanshaveextensivesupercooling chilling significantlyenhancescoldtolerance(Leeetal.,1987). acclimation responseinwhichbrief(minutestohours)exposure 1)(Worland andConvey, 2001),an cold-hardening (RCH;Fig. mites ( seasonal regulationofsupercoolingpoints,summer-acclimated activity bothon-andoff-host (LeeandBaust,1987).Inadditionto tolerate hightemperatures(>25°C),perhapstopermitsurvivaland capacity similartothatofmites(approximately–30°C),butcanalso Among thetrueinsectsonAntarctica,therearenotablydifferent Like mites,allAntarcticcollembolansstudiedthusfararefreeze- − − 15°C forpupaeandadults(Shimadaetal.,1991).Also,lower 9°C forpupae),makingitsignificantlylesscoldhardythan A. antarcticus − 30°C, whichisaccomplishedinpartbyaccumulationof The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 − Alaskozetes arcticus E. murphyi,arebothfreeze-tolerant,andin factare 20°C (Baustand Lee,1987;Worland, 2010), which

1) (seeLeeandDenlinger, 2010). and and P. steinenii has notbeenexamined.Nonetheless,asan Halozetes belgicae E. murphyi − 20°C inearlysummerthatincreases is likelybuffered fromextremesin are only− Ixodes uriae has awintersupercoolingpoint Styerotydeus mollis are freeze-tolerantdownto − 3°C forsummerlarvae ) arecapableofrapid 30°C insomespecies − sensu stricto has asupercooling 40°C (Sinclairand − 7°C forlarvae B. antarctica P. steinenii , hasa , which − 20°C, 85

The Journal of Experimental Biology Isolated tissuesof first reportofRCHinafreeze-tolerantinsect(Leeetal.,2006). illustrates thatRCHhasnoeffect onSCPsin (rank sumtest,P 86 REVIEW are chill-tolerant downtotheirsupercooling points (Worland and in mitesandCollembola(Fig. Antarctic insectsinvolvesadifferent mechanismthanthatobserved during hardening(Everattetal., 2012).Asdiscussedabove,RCHin in can occurinafrozenstate(Lee et al.,2006;Teets etal.,2008), RCH Antarctic arthropods.Interestingly, whereasRCHin rapid enhancementofcoldtoleranceisacommonadaptation in characterized in RCH inthisspecies(Teets et al.,2008).RCHhasbeensubsequently evidence indicatesaroleofcalciumsignalinginmediating hormones (Teets etal., 2008). Additionally, pharmacological implying thatRCHdoesnotrequireinputfromthebrain or they remainfreeze-tolerantyear-round (BaustandEdwards, 1979). of bothspeciesundergo seasonal coldacclimation,butitappears in maritimeAntarctica(Worland, 2010;Everattetal.,2012).Larvae colder climatesiswhatallowedthisspeciestosuccessfullyestablish cold tolerancesomewhatsurprising,butthis‘pre-’ to rarely experiencestemperaturesbelow experienced (BaustandLee,1981).Initsnativerange, is considerablylowerthantheminimummicrohabitattemperatures while were directlyexposedtothetesttemperature.InD,adiscriminatingtemperatureof Everatt etal.(Everattal.,2012)forD.InC,1 freezing withnoeffect onSCPs.DataaretakenfromWorland andConvey(Worland andConvey, 2001)forA etal.,2006)forC,and andB,Leeetal.(Lee In contrast,RCHintheAntarcticmidges(C) Cryptpygus antarcticus Fig. Recently, RCHwasdescribedin E. murphyi

.Rapidcold-hardening(RCH)inAntarctic arthropods. 1. − 5°C for1 h andgradualcoolingat2°Csuccessfullyenhancedfreezetolerance <0.05); inCandD,anasteriskindicatesasignificantimprovementsurvivaltheRCHgroups(ANOVA, Tukey, is onlyeffective whenlarvaeremainsupercooled E. murphyi Survival (%) Supercooling point (°C) is manifested by diurnal shifts in supercooling points (SCPs), as individuals sampled during cooler parts of the day have a lower SCP.is manifestedbydiurnalshiftsinsupercoolingpoints(SCPs),asindividualssampledduringcoolerpartsofthedayhavealower B. antarctica 100 –30 –28 –26 –24 –22 –20 –18 80 60 20 40 0 C A 12 14 Belgica antarctica –10 SCP (°C) Alaskozetes antarcticus –8 –6 –4 –2 –10°C/24 h (Everatt etal.,2012),suggestingthat

Control (4°C) 16 182022002040608

1). AntarcticmitesandCollembola (–5°C/1h) RCH are capableofRCH B. antarctica Time ofday(h) Belgica antarctica − B. antarctica 1.5°C, makingthislevelof

h at− –15°C/24 h * 5°C wasusedtoinduceRCHpriorfreezingattheindicatedtesttemperature,while‘control’ samples , andthiswasthe .

B. antarctica 10 1214 and (D) –20°C/24 h RCH in(A)theAntarcticmite E. murphyi Control RCH * ex vivo ex vivo Eretmoptera murphyi –4 –2 0 2 4 6 8 10 12 , Environmental temperature (°C) microhabitats to maintainwaterbalance.However, how these two gerlachei from thesamehabitat, environment (Benoitetal.,2008). Incontrast,twopredatorymites waterproofing hydrocarbonsthat limitevaporativewaterlosstothe antarcticus Antarctic mites,bothstrategies seemtobeinplay. Forexample, reduce waterlossorbeabletotoleratecellulardehydration.Among to reducewaterlossormechanismstolerateadehydratedstate. extremely tolerantofdesiccatingconditions,witheithermechanisms 2003). Thus,notsurprisingly, Antarcticarthropodsaretypically arthropods asawholearesusceptibletowaterloss(Gibbset al., their smallbodysizeandhighsurfaceareatovolume ratio, very littleannualprecipitation(Kennedy, 1993).Also,becauseof therefore unavailableformuchoftheyear, andinlandareasreceive biggest challengeforAntarcticarthropods.Water isfrozenand and fitnessofAntarcticarthropods,wateravailabilityisperhaps the the supercoolingpoint. RCH improvesthelowerlimitoffreezetolerancewithoutaffecting freezing wellbelowthesupercoolingpoint,andinthesespecies supercooling point.However, Convey, 2001),soRCHisachievedbyrapidlydecreasingthe

− Survival (%) Supercooling point (°C) To survivedesiccatingconditions, arthropodsneedtoeither While lowtemperatureisasignificantstresslimitingtherange 12.5°C for8 100 –25 –15 –20 –10 –5 80 60 20 40 0 0 − B D 12.5°C. InB,anasteriskindicatesasignificantdifference inSCP Cryptopygus antarcticus Eretmoptera murphyi , havehightranspirationrates andthusseekmoist involves physiologicalprotectionagainstthedamagingeffects of Habitat temp.~11°C Untreated

Alaskozetes antarcticus control The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 Time=17:00 h relies onwaterconservation, with athickcoatingof

h wasused.Exposureto0°Cfor1 Directly frozen Hydrogamasellus antarcticus 0°C/1 h Habitat temp.~2°C

Time=14:00 h B. antarctica 5C1hGradualcooling –5°C/1 h and (B)theAntarcticcollembolan * * (0.2°C/min) * P

h failedtoinduceRCH, and <0.05). TheinsetinC E. murphyi and Rhagidia survive A.

The Journal of Experimental Biology of et al.,2007b;Hayward2007).Inresponsetodesiccation,larvae larvae of readily surviveupwardsof50%waterloss(Worland, 2010),while dehydration. Atecologicallyrelevanthumidities,larvaeof antarctica state duringtheAntarcticwinter. of toleratingextremedesiccation,survivinginanear-anhydrobiotic Antarctic collembolanshavehighwaterlossrates,theyarecapable relevant conditions(Elnitskyetal.,2008a).Thus,ingeneral,while more than50%surviveda60%lossofbodywateratecologically must beabletosurviveinaseverelydehydratedcondition.Indeed, melting pointmatches theenvironmentaltemperature. dehydration arthropodsareneither frozennorsupercooled,astheir with freezetoleranceand avoidance),asduringcryoprotective now consideredbysometobe a thirdoverwinteringstrategy(along dehydration (Whartonetal.,2005). Cryoprotectivedehydrationis Antarctica, includingnematodes, arealsocapableofcryoprotective et al.,2008b).Somenon-arthropod soil-dwellingorganisms in 2003; Elnitskyetal.,2008a)andthemidge demonstrated inthecollembolan Among Antarcticspecies,cryoprotectivedehydrationhas been dehydration toleranceandtheabilitytoresistinoculativefreezing. cryoprotective dehydrationareapermeablecuticle,extreme temperatures (Holmstrupetal.,2002).Theprerequisites for thereby allowingthebodyfluidmeltingpointtotrackenvironmental gradient drawswateroutofthebodyintosurrounding ice, cuticles aresurroundedbyenvironmentalice,avaporpressure 2).Whenarthropods withwater-permeable dehydration (Fig. are capableofadistinctformdehydrationtermedcryoprotective 1000 in seawater, buttheyarefullycapableofsurvivingupto10 to thesea,somelarvaeof other formsofosmoticstress.Forexample,becausetheirproximity 2007a). Inadditiontodehydration,Antarcticarthropodsexperience habitats andsuccumbtoa30%lossofbodywater(Benoitetal., only activeforabriefperiodduringthesummer, requiremoist dehydration toleranceisrestrictedtothelarvalstage;adults,whichare cycles burn67%oftheircarbohydrateenergy stores.In energetically costly, aslarvaeexposedtofivedehydration/rehydration (Teets etal.,2012a).Also,fluctuatingmoistureregimesare near 100%,althoughsignificantmortalityoccursafterfivesuchcycles dehydration (resultingin~40%waterloss)and24 fluctuating moistureregimes.Survivalfollowingfourcyclesof24 cuticle (Benoitetal.,2007b).Larvaearealsohighlytolerantof clustering andbyincreasingthewaterproofingpropertiesoftheir 2008a). Because relies onliquidwatertomaintainbalance(Elnitskyetal., balance atanyrelativehumiditybelowsaturation,andtherefore detail, revealingthatthisspeciesisincapableofmaintainingwater Recently, thewaterbalanceof from 9to25%oftheirbodywaterperhour, dependingonspecies. At 5%relativehumidity(RH)and0°C,collembolanslostanywhere significantly higherratesofwaterlossthananythemitespecies. Block (Worland andBlock,1986)foundthatcollembolanshad seven speciesofAntarcticmitesandcollembolans,Worland and and exhibitverylittleresistancetowaterloss.Inacomparisonof has notbeenexamined. mites toleratewinterdesiccation,whenliquidwaterisunavailable, REVIEW Like thecollembolans,Antarcticmidges Recently, ithasbeendiscoveredthatmanyAntarcticarthropods Antarctic collembolansaretypicallyfoundinmoistenvironments B. antarctica mOsm seawater(Elnitskyetal.,2009). B. antarctica lose waterveryrapidlybutareextremelytolerantof C. antarcticus combat highwaterlossratesbybehaviorally survive upto70%lossofbodywater(Benoit B. antarctica C. antarcticus is unabletopreventdehydration,it C. antarcticus are alsoatriskofimmersion B. antarctica was examinedinmore (Worland andBlock, E. murphyi

h rehydrationis B. antarctica E. murphyi (Elnitsky

days in and B.

h , placed inthepresenceoficeat temperatures inthepresenceofice.InA,whenafullyhydratedspringtailis antarcticus cryoprotective dehydration.(B)Water contentandosmolalitydatafrom (Elnitsky etal.,2008a).(A)Illustrationoftheunderlyingphysics Antarctic collembolan arthropods. Fig. freezing atecologically relevantsoilmoisture levels(Elnitskyet in afieldsetting,larvaeare capableofavoidinginoculative cryoprotective dehydrationin dehydration although theabilityoftardigrades toundergo cryoprotective temperature inbothfrozenand dehydratedstates(Sømme,1996), Antarctic tardigradesalsoappear tobecapableofsurvivinglow likely tobeableavoidinoculativefreezinginthe field. considered alaboratoryartifact,becauseneitherofthesespecies is enchytraeid worm(PedersenandHolmstrup,2003)but was in afreeze-tolerantnematode(Whartonetal.,2003)and an subzero temperatures.Cryoprotectivedehydrationwasdescribed intolerant species,asitallowsarthropodstoremainunfrozen at dehydration wasoriginallyconsideredanadaptationforfreeze- cryoprotective dehydrationhasbeendemonstrated.Cryoprotective freeze-tolerant. Also, dehydration wassomewhatsurprising,becausethisspecies is point; DM,drymass. which reducestheriskofinternalfreezing.WC,watercontent;MP, melting melting temperatureandwaterpotentialapproachthatofthesurroundingice, water contentdecreaseswhileosmolalityincreases,suchthatthebodyfluid drives wateroutoftheinsectintosurroundingice.After28 inside thebodyisconsiderablyhigherthanthatofsurroundingice,which B In thecaseof A

.Schematicillustrationofcryoprotective dehydrationinAntarctic 2. –1 Water content (g H2O g DM) 1.8 1.6 1.4 1.2 2.6 2.0 2.4 2.2 Ψ=–9.7 bar Body fluidMP=–0.8°C Body fluidosmolality=424mOsm WC=2.3 gH Springtail: Initial Ψ=–36.2 bar T=–3.0°C Surrounding ice: over thecourseofa28-dayexposuretograduallydecreasing This exampledemonstratescryoprotectivedehydrationinthe The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 Water content Osmolality sensu stricto –0.6 0 H 2 B. antarctica O g 2 Cryptopygus antarcticus O –3.0 –1

B. antarctica 5 101520 DM Temperature (°C) –3.0 Time (days) − 3°C, thewaterpotential( B. antarctica has notbeenexamined.While , thediscoveryofcryoprotective is thefirsttrueinsectinwhich Ψ=–26.6 bar Body fluidMP=–2.2°C Body fluidosmolality=1181mOsm WC =1.4gH Springtail: After 28daysacclimation , usingdatafromElnitskyetal. –3.0 Ψ=–36.2 bar T=–3.0°C Surrounding ice: has yettobeexamined –3.0

25 2 O g Ψ –1 ; 1

–3.0 DM 30

days, the bar=100 1000 1200 1400 800 600 200 400 C.

kPa) Body fluid osmolality (mOsm) 87

The Journal of Experimental Biology 88 REVIEW supercooling points (<–15°C)werecomparedwith thosewith‘high’ microarray studiesofcoldtolerance. Inthefirst,animalswith‘low’ 1. Table responsive genesidentifiedin Antarctic arthropodsisprovidedin the world’s mostextremearthropods.A summaryofthestress- provide cluesaboutthemechanisms ofstresstoleranceinsome conducted intwospecies,thecollembolan technology, althoughmolecularexperimentshaveonlybeen benefited fromadvancesinmolecularbiologyand‘omics’ extreme dehydrationinAntarcticarthropodsaswell. 2007b; Elnitskyetal.,2008b)haveimplicateditsimportanceduring antarcticus capable ofanhydrobiosis(Clegg,2001).Recentstudies in the bloodsugarandischiefosmoprotectantinarthropods (Elbein etal.,2003).Inarthropods,trehalosetypicallyfunctions as demonstrated toaccumulatetrehaloseinresponsedesiccation certain bacteria,fungi,plantsandmetazoans,havebeen osmoprotectant duringdehydration.A rangeoforganisms, including desiccation-tolerant organisms istheimportanceoftrehaloseasan theme thatisemerging fromstudiesofbothpolarandtropical osmoprotectants (LeeandBaust,1981;BaustLee,1983).One instead reliesprimarilyonglucose,erythritolandtrehaloseas midge response todesiccation(Elnitskyetal.,2008a).Incontrast,the chief cryoprotectantandalsoaccumulatesglucosetrehalosein 1995). Likewise,thecollembolan accumulating levelsupwardsof0.5 A. antarcticus type andamountvaryfromspeciestospecies.Forexample,themite accumulates somesortofosmoprotectivecompound,althoughthe hallmark ofAntarcticarthropods.Everyspeciesprofiledthusfar accumulation oflow-molecular-weight osmoprotectantsisa arthropods andhighlightsomeavenuesforfutureresearch. physiological mechanismsofstresstoleranceinAntarcticterrestrial on advancesinmolecularbiology. Here,wewillreviewthe markers ofenvironmentalstress,andrecentstudieshavecapitalized Nonetheless, earlystudiesinthe1980scharacterizedbiochemical stress toleranceinAntarcticarthropodshavereceivedlittleattention. In comparisonwithtemperateinsects,themolecularmechanismsof energetic demandsbeyondthatoflowtemperaturealone. associated withfreezingimposesadditionalcellularstressand repeated diurnalcoldcycles.Thus,thecellulardehydration 2011), andsimilarresultsareobservedwhenlarvaeexposedto energy reservesthantheirsupercooledcounterparts(Teets etal., approximately sevenfoldhighermortalityandhave~20%less − supercooled atsubzerotemperatures.Followinga60 evidence indicatesthatitispreferableforlarvaetoremain supercooled, providedlarvaeavoidinoculativefreezing.Recent subzero temperatureexposureslarvaearecapableofremaining typical microhabitattemperatures,meaningthatduringbrief supercooling pointof dehydration maybeusedwithinasinglespecies.Furthermore,the arthropods wherebothfreezetoleranceandcryoprotective al., 2008b).Thus,B.antarctica in Antarctic arthropods in Antarctic tolerance stress Biochemical andmolecularmechanisms of 5°C, larvaethatareinoculativelyfrozenexperience Molecular experimentsin In recentyears,physiologicalstudiesofAntarcticarthropodshave Like theirtemperatecounterparts,seasonalandstress-induced B. antarctica B. antarctica (Elnitsky etal.,2008a)and primarily usesglycerolasanosmoprotectant, . Regardless,thesestudiesarebeginningto accumulates verylowlevelsofglyceroland B. antarctica C. antarcticus represents auniquecaseamong C. antarcticus

larvae ( mol B. antarctica l –1 C. antarcticus are restrictedtotwo (Block andConvey, − 7°C) islowerthan uses glycerolasits

(Benoit etal., h exposureto and the C. water movement duringstressfulconditions such asdehydration insects (Lalouetteetal.,2011). common inAntarctica,areknown tocauseoxidativedamagein Furthermore, repeatedboutsof freeze–thawexposure,whichare result ofozonedamage(Weatherhead andAndersen, 2006). radiation (LiaoandFrederick, 2005),whichisintensifyingasa arthropods, asAntarcticsunlightcontainsveryhighlevelsof UV of mates.ResistancetooxidativedamageiscrucialforAntarctic constant exposuretosunlightastheywalkonthesurfaceinsearch levels ofantioxidantcapacity, probablybecauseoftheirnear- Martinez etal.,2008).Adultsof than thatofatemperatefreeze-tolerantinsect, the antioxidantcapacityof extremely highresistancetooxidativedamagein after exposuretosunlight.Indeed,expressionofthesegenesconfers encoding catalaseandthreeheatshockproteins,modestlyincrease Superoxide dismutasemRNA levels,aswellthemRNAs absence ofovertoxidativestress(Lopez-Martinezetal.,2008). antioxidant enzymesuperoxidedismutaseathighlevelseveninthe shock .Likewise,larvaeexpressgenesencodingthe feeding andgrowing. produce heatshockproteinsathighlevelsevenwhiletheyare Feder, 1997),larvaeofB.antarctica proteins typicallyhindersgrowthanddevelopment(Krebs maritime Antarctica.Whereashighexpressionofheatshock environmental stress,whichcanbefrequentandunpredictablein heat shockproteinslikelyprovidesyear-round protectionagainst (small hsp,hsp70andhsp90)inlarvae.Thisconstantpresenceof nor coldincreasedexpressionofthreedifferent heatshockproteins antarctica levels allthetime(Rinehartetal.,2006).Whileadultsof constitutively expressgenesencodingheatshockproteinsathigh very lowlevelsuntiltheproteinsareneeded,larvaeof whereas mostinsectsexpressgenesencodingheatshockproteinsat important mediatorsofstresstolerancein studies. Asintemperateinsects,theheatshockproteinsare antarctica matrix proteintenebrinwasverifiedtobedownregulated. upregulated byqPCR,whileanmRNA encodingtheextracellular 49Ah andchitin-bindingperitrophinA)wereconfirmedtobe (endocuticle structuralglycoproteinSgAbd-4,cuticularprotein collembolans (Burnsetal.,2010).Inthisstudy, threegenes involved inthemoltcyclewereupregulatedcold-acclimated 5400 ESTs), andonceagain,anumberofcuticulargenes experiment wasconductedlaterwithalarger microarray(containing checkpoint homologinvolvedincellcycleregulation.A similar confirmed byqPCRincludeacuticularproteinandCHK1, component oflowtemperaturesurvival.Specificupregulatedgenes synthesis, suggestingthatboostingenergy productionmaybea group includeseveralmitochondrialgenesinvolvedinATP Other genesupregulatedinthe‘low’ grouprelativetothe‘high’ capacity inAntarcticcollembolans(Worland andConvey, 2008). importance ofthecuticleandmoltcycleinregulatingsupercooling other structuralconstituentsinthe‘low’ group,confirmingthe patterns indicateupregulationofanumbercuticularproteinsand (ESTs), andthuswasnotcomprehensive.Nonetheless,expression This microarraycontainedasubsetof672expressedsequencetags responsible forloweringsupercoolingpoints(Pura supercooling points(>–15°C),todeterminewhichgenesare Recently, aquaporinshavebeenimplicatedaskeyregulators of Constitutive defensesin Relative tootherAntarcticarthropods,themidge have atypicalheatshockproteinresponse,neither has beensubjectedtothelargest numberofmolecular The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 B. antarctica B. antarctica B. antarctica are abletocircumventthisand larvae isfivetimesgreater are notrestrictedtoheat B. antarctica E. solidaginis have evenhigher B. antarctica ć etal.,2008). B. antarctica . However, (Lopez- , as B. B.

The Journal of Experimental Biology aquaporins found immunoreactivitytofour different aquaporin mediating stresstolerance.A secondstudyof dehydration, soitisunclearwhat, ifany, rolethisgeneplays in However, mRNA expressiondidnotchange inresponseto that itmayplayageneralrole inwatermovementacrosscells. aquaporin geneisexpressedin severaldifferent tissues,indicating not ureaorglycerol,across the cellmembrane.Thisspecific Xenopus aquaporin-1 likegenefrom characterized thefirstaquaporinfromanAntarcticarthropod, an mediating stresstolerance.Gotoetal.(Gotoal.,2011) clonedand immersion inseawater, aquaporinslikely play animportantrolein forms ofosmoticstress,includingfreezing,dehydration and 1999). BecauseAntarcticarthropodsarechallengedbynumerous sometimes othersolutes,acrossthecellmembrane(Borgnia etal., 2010). Aquaporinsarepore-formingproteinsthatcarrywater, and (Liu etal.,2011) andfreezing(Philipetal.,2008;PhilipLee, REVIEW Species: Only genesconfirmedwithatargetedapproach(i.e. A Structural Functional category Table 1.Listofstress-upregulatedgene Other Cell death/longevity Cryoprotectant mobilization Lipid modification Detoxifcation Heat shockproteins

ntioxidant enzymes Ca oocytes, thisproteiniscapableoftransmittingwater, but , Cryptopygusantarcticus Ba Ba Ba Ba Ba Ba Ba Ca Ba Ba Ca Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ca Ca Ba Ba Ba a cuticular protein Ca pce Gene Species B. antarctica ; Ba , Belgicaantarctica icfne rti D Lopez-Martinezetal.,2009 D zinc fingerprotein relish D " " " " H,C,D D D " phosphoenolpyruvate carboxykinase S,D phospholipase A2activatingprotein metallothionein 2 superoxide dismutase 70 Burnsetal.,2010 C endocuticle structuralglycoproteinSgAbd-4 eti D Teetsetal.,2012b " Purac etal.,2008 D " D C " " " D " vacuolar (H pacifastin-related serineproteaseinhibitor " " D D D CHK1 checkpointhomologue spermidine synthase Teetsetal.,2013 thread D D " sestrin D D pyrroline-5-carboxylate reductase H,C,D glycerol-3-phosphate dehydrogenase Lopez-Martinezetal.,2009 aldehyde/ketone reductase transporter1 trehalose-6-phosphate phosphatase D trehalose-6-phosphate synthase " -6-phosphatase D trehalase C,D D " glycogen phosphorylase fatty acylCoA Lopez-Martinezetal.,2009 fatty aciddelta cytochrome P4506a23 UDP-glycosyltransferase D " " " Ri D cytochrome P45028a5 Teets " " H,A,S,D D D C C et small heatshockprotein myosin light-chainkinase actin muscle-specific actin chitin-binding peritrophinA al., cuticular protein49Ah 2012b 40 90 catalase s identifiedinAntarcticarthropods

kDa heatshockprotein kDa heatshockprotein kDa heatshockprotein . Whenexpressedin northern blotorqPCR)ar + ) ATPase  B. antarctica eauae " D 9 desaturase . Typeofstress:C,cold;D,dehydration; eauae Lopez-Martinezetal., 2009 D 9 desaturase e includedinthetable. transcripts encoding glycogenphosphorylase and upregulation ofgenesinvolved inglucosemobilization,including proline synthesis.Highand lowtemperatureinducesrapid breakdown, gluconeogenesis,polyol andtrehalosemetabolism, al., 2013)profiledtheexpression of11 genesinvolvedinglycogen stress responseinB.antarctica of osmoprotectantsalsoappear tobeessentialcomponentsofthe also playacrucialroleinmediatingdehydrationstress. reduced thewaterlossofmidguttissue,suggestingthataquaporins water redistributionduringfreezing.Additionally, mercuricchloride Malpighian tubuletissue,indicatingthataquaporinsarecrucial for reduced the blocking aquaporinspharmacologicallywithmercuricchloride aquaporin geneshasnotbeenestablished.Inthesamestudy, inducible (Yi etal.,2011). However, thesequenceidentityofthese antibodies fromdifferent species, andsomeofthesewerestress Genes involvedinmobilizationofenergy reservesandsynthesis The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 D yeo tes Reference(s) Type ofstress D C A H,S,D Rinehartetal.,2006;Lopez-Martinez et H,A,S,D,C ex vivo ,S,D H,heat;A,anoxia;S,directsunlight. freezing toleranceoffatbody, midgutand Teets etal.,2012b " Purac etal.,2008 Rine Lopez-Martinez etal.,2008;Lopez- nehart etal.,2006;Lopez-Martinez et Teets etal.,2012b al., 2008;Lopez-Martinezet2009; Teets etal.,2011;2012b al., 2008;Lopez-Martinezet2009; al., 2008;Lopez-Martinezet2009 Martinez etal.,2009 . UsingqPCR,Teets etal.(Teets et hart etal.,2006;Lopez-Martinez et 89

The Journal of Experimental Biology 90 REVIEW understanding ofstresstolerancein during prolongeddehydration. 2007b; Elnitskyetal.,2008b)andproline(Teets etal.,2012b) synthesis, consistentwithaccumulationoftrehalose(Benoitetal., these treatmentsupregulategenesinvolvedintrehaloseandproline and cryoprotectivedehydrationalsoinduceexpressionof proline synthesis.Incontrast,whileslowdehydrationat98%RH concurrent downregulationofgenesinvolvedintrehaloseand mobilizing enzymes(i.e.PEPCKandglycogenphosphorylase)with heat andcold,namelyupregulationofgenesencodingglucose a similartranscriptionalsignatureasthatobservedinresponseto type ofdehydrationexperienced.Rapidat75%RHhas dehydration, geneexpressionpatternsarehighlydependentonthe involved intrehaloseandprolinesynthesis.Inresponseto and lowtemperatureresultinageneraldownregulationofgenes larvae of previous observationsofcold-inducedglucosemobilizationin enzyme ofgluconeogenesis.Theseresultsareconsistentwith phosphoenolpyruvate carboxykinase(PEPCK),therate-limiting suggesting that dehydrationcausesamolecular shiftto in glycolysis,thetricarboxylic acidcycleandlipidmetabolism, downregulation ofcentralmetabolic genes,includinggenesinvolved Desiccation andcryoprotective dehydrationalsocauseageneral prolonged periodsofcellular stress (Teets andDenlinger, 2013). pathway inhibitsapoptosisand otherformsofcelldeathduring particularly importantforsurviving theAntarcticwinter, asthis 3).We hypothesizethatautophagyis promoting cellsurvival(Fig. components duringdehydration,therebyconservingenergy and and autophagyfunctiontorecycleremovedamagedcellular upregulation ofheatshockproteins,ubiquitin-mediatedproteasome genes. Taken together, theseresultssuggestthatcoordinated including significantenrichmentofproteasomalandautophagy recycling/degradation ofproteinsandcellularmacromolecules, desiccation causedupregulationofgenesinvolvedin the upregulated byoneorbothdehydrationtreatments.Concurrently, antarctica role ofheatshockproteinsduringenvironmentalstress in being differentially expressed. Theseresultsconfirmedthecrucial dehydration resultedin~24and~18%,respectively, ofallgenes changes ingeneexpression,asdesiccationandcryoprotective and cryoprotectivedehydration.Bothtreatmentsresultinsweeping transcripts inresponsetobothdesiccationataconstanttemperature Teets etal.(Teets etal.,2012b)profiledtheexpression of~13,500 conducted inAntarcticarthropods.UsingIllumina-basedRNA-seq, dehydration. transport chaingenes,suggestingashutdownofmetabolismduring downregulated inresponsetodehydration,includingtwoelectron remodeling (Bayleyetal.,2001).Inaddition,severalgenesare cytoskeletal reorganization (Chenetal.,2005)andmembranelipid consistent withpreviousobservationsthatdehydrationcauses coding forcytoskeletalproteinsandmembranerestructuring, stress. Othergenesupregulatedduringdehydrationinclude denaturation andoxidativedamagearesymptomsofdehydration (superoxide dismutaseandcatalase),indicatingthatprotein hsp70 andhsp90)genesencodingtwoantioxidantenzymes genes includethoseencodingthreeheatshockproteins(hsp26, expressed eitherduringdehydrationorrehydration.Upregulated northern blotsconfirmedthat23ofthesewereindeeddifferentially al., 2009)obtainedanumberofdehydration-responsiveclones,and subtractive hybridization,Lopez-Martinezetal.(Lopez-Martinez Non-targeted, ‘omics’ approacheshavealsobenefitedour To date,asinglegenome-wideexpressionstudyhasbeen B. antarctica , as15different heatshock proteintranscriptswere (Teets etal.,2011). Incontrast,acute high B. antarctica . Usingsuppressive pepck, B. prolonged periodsofenvironmentalstress. damaged proteinsandorganelles,therebypromotingcellsurvivalduring These genes,inturn,carryoutessentialcellrecyclingfunctionstoremove upregulated anddownregulatedinresponsetodehydrationisincluded. genes, outlinedindashedlines.Intheboxes,numberofgenes damaging effects ofdehydrationcauseupregulationkeycellrecycling expression inlarvaeof Results aretakenfromanRNA-seqstudyofdehydration-inducedgene during extremedehydrationintheAntarcticmidge Fig. a handfulofspecies. Inthepast30 Antarctic arthropodcommunity isdepauperateandconsistsofonly In contrasttotheabundanceof arthropodsonothercontinents,the (Teets etal.,2012b). desiccation ataconstanttemperature andcryoprotectivedehydration three polyols(erythritol,sorbitolandmannitol)inresponseto both acids (proline,glutamineandlysine),asinglesugar(fructose) and dehydration inthisspeciesobservedaccumulationofthreeamino antarctica part explainthecross-tolerancebetweenthesetwostresses in including theosmoprotectantsglycerolanderythritol,whichmay in metabolites showsimilarresponsestocoldanddesiccation, metabolic adaptationstoheat,freezinganddehydration.Several metabolomics, Michaudetal.(Michaudal.,2008)profiled from recentadvancesinmetabolomics.Usingnon-targeted GC-MS understanding ofthestressphysiology proteins aresynthesizedwhileothersdegraded.Finally, our abundant duringdesiccation,indicatingthatcertaincontractile and myosin.Interestingly, severalcontractileproteinsarealsoless 13 arecellstructuralproteins,includingseveralisoformsofactin et al.,2009).Ofthe18proteinsmoreabundantduringdehydration, cell structuralrearrangementsareessentialduringdehydration(Li rehydration in metabolism governresponsestoextremeenvironmentalconditions. indicating thatcoordinatedchangesingeneexpressionand in themetabolomecorrelatewellwithgeneexpressionchanges, up ofmetabolicendproducts(Teets etal.,2012b).Indeed,changes hypometabolism thatconservesenergy andpreventsthetoxicbuild- Conclusions directions andfuture At theproteinlevel,aproteomicsstudyofdehydrationand Repair misfoldedproteins

.Flowchartillustratingtheimportance ofcellrecyclingpathways 3. 28 totalingenome 14 upregulated,5downregulated Heat shockproteins (Hayward etal.,2007).A secondmetabolomicsstudyof The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089490 B. antarctica Misfolded proteins B. antarctica 53 genestotalingenome 29 upregulated,0downregulated Ubiquitin-mediated proteasome Degradation ofdamagedproteins Desiccating conditions supported theideathatcytoskeletaland Osmotic stress (Teets etal.,2012b).Inthisdiagram,the

years, researchers havebeen 10 genestotalingenome 8 upregulated,0downregulated Autophagy and macromolecules Damaged organelles B. antarctica of damagedmacromolecules Removal andrecycling Belgica antarctica. has benefited B.

The Journal of Experimental Biology antarctica compared dehydration-inducedgeneexpressionchangesin mechanisms. Forexample,Teets etal.(Teets et al., 2012b) whether eachspeciesreliesonauniquesuiteofmolecular conserved molecularadaptationstoenvironmentalstress,or Antarctic specieswouldrevealwhethertherearecrucial, physiological genomicsofstressresponsesacrossmultiple take advantageofacomparativedesign.Comparative have notbeenanymolecularstudiesinAntarcticarthropodsthat and differences amongAntarcticspecies.However, todatethere successfully usedcomparativeapproachestoelucidatesimilarities water balanceofAntarcticmites(Benoitetal.,2008),have collembolans (Sinclairetal.,2003;Sinclair2006)andthe ecophysiological studies,suchasthecoldtoleranceofAntarctic would beanincreasedrelianceoncomparativephysiology. Basic promising starts,butthereisstillmuchtobelearned. dehydration inpolararthropods(Worland andBlock,2003)are (Rinehart etal.,2006)andtheprevalenceofcryoprotective constitutively highexpressionofheatshockproteinsin have beenelusivetophysiologists.Discoveriessuchasthe Antarctic arthropods,uniqueadaptations,iftheyexist, Thus, despiterecentadvancesinthemolecularphysiologyof al., 2011), havebeenpreviouslydescribedintemperatespecies. Lee, 1983)andtheroleofaquaporinsduringfreezetolerance(Yi et arthropods, suchasaccumulationofosmoprotectants(e.g.Baustand and temperatecounterparts.MostadaptationsdescribedinAntarctic adaptations thatdistinguishAntarcticarthropodsfromtheirtropical However, whatisstilllackinganunderstandingoftheunique advances inourknowledgeoftheworld’s mostextremearthropods. recent advancesinmolecularbiologyhavefosteredsignificant intently studyingthephysiologicalecologyofthesearthropods,and warming would increasesnowmelt,which wouldreduce addition todirecteffects oftemperatureandprecipitation, climate distribution ofterrestrialarthropods (BaleandHayward,2010).In precipitation events,whichcan furtherinfluencetherangeand predict anincreasedfrequency ofextremetemperatureand for reproduction(BaleandHayward, 2010).Climatemodelsalso increase metabolicrate,depleting energy reservesnormally reserved reduced overwinteringmortality, highertemperatureswouldalso arthropods remainstobeseen.Whilemilderwinterswouldsuggest However, whatthisrapidwarmingtrend meansforAntarctic higher inthedecade2000–2009thanitwasfrom1990to1999. (the coldestmonthoftheyear)atPalmerStationisafulldegree is largely manifestedinthe winter; themeanAugusttemperature average dailytemperatureof~0.1°Cevery2 20 al., 2009).Forexample,atPalmerStation,overashortperiod of rapid warmingratesontheplanetoverlast50 Antarctica’s arthropoddiversity, isexperiencingoneofthemost climate. TheAntarcticPeninsula,whichishometomuch of arthropods isparticularlyimportantinthefaceofachanging molecular underpinningsofthesedifferences. (see Convey, 2010),andtoolsarenowavailabletorevealthe life historydifferences betweenAntarcticandtemperatespecies to stressare‘Antarcticspecific’.Thereclearphysiologicaland related temperatespecies,toidentifywhichmolecularadaptations could alsobeconductedbetweenAntarcticarthropodsandclosely stress, despiteoccupyingsimilarmicrohabitats.Suchcomparisons rely ondistinctmolecularadaptationstocombatdehydration arctica REVIEW One waytopotentiallyuncoveruniqueAntarcticadaptations Understanding theenvironmentalphysiologyofAntarctic years from1990to2010,therewasasteady, consistentrisein (Clark etal.,2009),andfoundthatthesetwoarthropods with thoseofanArcticcollembolan,

er Fg 4).Warming years (Fig.

years (Turner et Megaphorura B. antarctica B. eot .B,LpzMrie,G,Mcad .R,Entk,M . e,R . Jr R. E., Lee, M.A., Elnitsky, M. R., Michaud, G., Lopez-Martinez, J.B., Benoit, as,J .adLe R.E.,Jr Lee, and J.G. Baust, eot .B,LpzMrie,G,Entk,M . e,R . radDnigr D. Denlinger, and R.E.,Jr Lee, M.A., Elnitsky, G., Lopez-Martinez, J.B., Benoit, next 364 each daywascalculatedbytakingthemeantemperatureforthatplus noise andhighlightgeneraltrendsintemperature.A yearlymovingaveragefor das/lter/index.jsp). Dataweretransformedwithamovingaveragetoreduce Ecological ResearchNetworkdatarepository(https://metacat.lternet.edu/ from 1January1990to2010wereobtainedtheUSLongTerm Antarctic Peninsulaillustratingwarmingtrends. Fig. (OPP-ANT-0837613). This workwassupportedinpartbyagrantfromtheNationalScienceFoundation the figures. N.M.T. andD.L.D.conceivedthemanuscriptwrotepaper. N.M.T. prepared The authorsdeclarenocompetingfinancialinterests. We appreciatethehelpful commentsprovidedbytwoanonymousreviewers. ranges andpopulationdynamicsofAntarcticarthropods. to perturbationsintheenvironmentisessentialforforecastingfuture al., 2011). Thus,understandingtheplasticityofAntarcticarthropods to snowmeltcouldincreasetheriskofinoculativefreezing(Teets et freeze–thaw cyclesexperienced.Also,increasedsoilmoisturedue microhabitat thermalbuffering, therebyincreasingthenumberof ae .S n awr,S.A.L. V. Hayward, and Sbordoni, J.S. Bale, and P. Convey, G., Carchini, G., Allegrucci, as,J .adEwrs J.S. Edwards, and J.G. Baust, aly . eesn .O,Kig,T,Khe,H .adHlsrp M. Holmstrup, and H.R. Köhler, T., Knigge, S.O., Petersen, M., Bayley, R.E.,Jr Lee, and J.G. Baust, References Funding Author contributions Competing interests Acknowledgements as,J .adLe R.E.,Jr Lee, and J.G. Baust, antarctica the Antarcticmidge, D.L. Denlinger, and J. Exp.Biol. terrestrial arthropod: cryoprotectant accumulationpatternsinanAntarcticinsect. L. J. InsectPhysiol. Drought acclimationconferscoldtolerance inthesoilcollembolan Antarctic andsub-Antarcticislands. geographic relationshipsamongorthocladinechironomidmidgesfrommaritime . Antarct.J.US antarctic midge,

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The Journal of Experimental Biology Hogg, D.L. Denlinger, and Jr R.E., Lee, L.H., Sandro, J.P., Rinehart, S.A.L., Hayward, oo .G,Pii,B . et,N . aaaai . e,R . radDenlinger, and Jr R.E., Lee, Y., Kawarasaki, N.M., Teets, B.N., Philip, S.G., Goto, L.M. Matzkin, and F. Fukuzato, A.G., Gibbs, vrt,M . oln,M . ae .S,Cne,P n awr,S.A.L. Hayward, and P. Convey, J.S., Bale, M.R., Worland, M.J., Everatt, lisy .A,Bni,J . oe-atnz . elne,D .adLe R.E., Lee, and D.L. Denlinger, G., Lopez-Martinez, J.B., Benoit, M.A., Elnitsky, D. Carroll, and I. Pastuszak, Y. T., Pan, A.D., Elbein, lisy .A,Hyad .A . ieat .P,Dnigr .L n e,R.E., Lee, and D.L. Denlinger, J.P., Rinehart, S.A.L., Hayward, M.A., Elnitsky, 92 REVIEW resae P. Greenslade, lg,J.S. Clegg, ai,R.C. Davis, P. Convey, P. Convey, Purać M.A.S., Thorne, M.S., Clark, lisy .A,Bni,J . elne,D .adLe .E,Jr R.E., Lee, and D.L. Denlinger, J.B., Benoit, M.A., Elnitsky, P. J.A., Pugh, D.A., Hodgson, C.-D., Hillenbrand, J.A.E., Gibson, P., Convey, P. Convey, P. 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