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*Author ([email protected]) for correspondence , DC20036,USA. Grove, CA93950,USA. © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,265-275doi:10.1242/jeb.114157 Received 17September 2014;Accepted24November 2014 1 (Nigmatullin etal.,2001). predators arespermwhales,sharksandlarge teleostfishes in thenorthernhemisphere(Fieldetal.,2013).Itsmostcommon also preyonmuchlarger demersal ,particularlyathigherlatitudes al., 2001;MarkaidaandSosa-Nishizaki,2003),butlarger individuals , krill,pteropodsandmesopelagicsquids(Nigmatullin et mesopelagic micronektonthroughoutlife,particularlymyctophid and EquatorialUpwellingZone. the CaliforniaCurrent,(Humboldt)CostaRicaDome and preyisassociatedwithhighlyproductiveoceanicsystems – southern .Itisanecologicallyimportantspeciesasbothpredator witharangeextendingfromBritishColumbia to or jumboflyingsquid,isalarge pelagicsquidofthefamily Dosidicus gigas Flickering, Crypsis KEY WORDS:,,Flashing,Signaling, two dynamicdisplayswithoutartificiallightingatdepthsofupto70 focus oncolor-generating(chromogenic)behaviors.We documented behaviors infree-swimming National Geographic’s Crittercam,wewereabletoobservenatural oceanic environment.Byusingananimal-bornevideopackage, behaviors becauseofdifficulties instudyingthisactivepredatorits and ecologicallyinfluentialspecies,yetlittleisknownaboutitsnatural Dosidicus gigas Hannah Rosen studied insitu Chromogenic behaviorsoftheHumboldtsquid( RESEARCH ARTICLE with otherspecies. not beendescribedinothersquid,functionalsimilaritiesareevident under inhibitoryneuralcontrol.Althoughflashingandflickeringhave in flickering,oftenbeforeaflashingepisode,indicatethatflickeringis may provideadynamictypeofcamouflage.Rapidandglobalpauses down-welled lightinthewatercolumn,suggestingthatthisbehavior , andtheresultingpatternsmimicreflectionsof by irregularwave-likeactivityinneighboringpatchesof whenever flashingwasnotoccurring.Thisbehaviorischaracterized altered. Anotherdynamicdisplaytermed‘flickering’ wasobserved and thephaserelationshipwithanothersquidcanalsoberapidly signaling. Amplitudeandfrequencyofflashingcanbemodulated, video frame,andthisbehaviorpresumablyrepresentsintraspecific was almostalwaysobservedwhenothersquidwerevisibleinthe oscillation (2–4 One dynamicpattern,termed‘flashing’ ischaracterizedbyaglobal INTRODUCTION ABSTRACT Hopkins Marine Station of Stanford University,Hopkins MarineStation of 120Oceanview Blvd, Pacific

Hz) ofbodycolorbetweenwhiteandred.Flashing d’Orbigny 1835,commonlyknownastheHumboldt (Humboldt orjumboflyingsquid)isaneconomically 1, 2 *, William Gilly National GeographicSociety, Remote Imaging, D. gigas D. gigas with ananimal-bornevideopackage is frequentlyassociatedwiththe D. gigas in theGulfofCaliforniawitha 1 , LaurenBell feeds primarilyon 1 , KylerAbernathy

m. chromogenic behaviorof chromatophores (Packard,2011). We areunawareofanywork on et al.,2012)utilized ananimal-bornevideoand instrumentpackage, published inthescientificliterature. colors, oceanicsquidsuchas loliginid ,whichgenerally havechromatophoresofseveral and deimaticdisplays(HanlonMessenger, 1996).Unlike appear tobeprimarilyrelatedcamouflage,intraspecificsignaling (Bush etal.,2009).Inloliginidsquidchangesinspatialpatterning coastal orshelfenvironmentsandonafewmesopelagicspecies entirely onseveralspeciesinthefamilyLoliginidaethatinhabit color-changing (chromogenic)behaviorsinsquidis basedalmost brain (BullockandHorridge,1965).Ourknowledgeofnatural by motorneuronswithcellbodiesinthechromatophorelobes of the fibers (seeFlorey, 1969).Thesemusclefibersaredirectlyinnervated visible toanobserver, throughtheactionof aringofradialmuscle of anelasticpigmentsacthatcanbeexpanded,andthereby made neuromuscular chromatophores.Thesesmallorgans arecomposed ) istheabilitytochangeskincolorthroughuseof generated noisecanskewbehaviorsofsquidinunknownways. Vechionne etal.,2002),butbrightilluminationandvehicle- mesopelagic depths(HuntandSeibel,2000;Kuboderaetal.,2007; manned submersiblesprovideunique Observations madewithremotelyoperatedvehicles(ROVs)or nuances ofbehaviorthatacousticmethodscannotresolve. information onsocialstructureandschooling,buttherearemany night (Benoit-BirdandGilly, 2012),thusprovidingnon-invasive Acoustic samplingsuggeststhat movements, e.g.foraging,predatorevasionorsocialinteractions. but thesedevicesdonotrevealthebehavioralcontextfor gigas about naturalverticalmovementsandswimmingbehaviorsof 2006; Davisetal.,2007). throughout thewatercolumnarecommonatallhours(Gillyetal., several hundredmeters(Stewartetal.,2012).Rapidexcursions 10 to50 typicallyspendsthemajorityofnight-timehoursatdepths species (andotherommastrephids)complicatesuchstudies.This and theoceanicdistributionlargely mesopelagichabitatofthis relatively littleisknownaboutitsnaturalhistoryorsocialbehaviors, world’s twelfthlargest single-speciesfishery(FAO, 2012). gigas associated withthisenvironmentalfeature(Stewartetal.,2014). appears tobedirectlyrelatedanabundanceofmyctophids presence oftheHumboldtsquidinornearOMZoff hostile tomostlarge marineanimalsthatarenotair-breathers. The oxygen minimumzone(OMZ),ahypoxicmesopelagicregionthatis A previous studyofverticalmovementsDosidicusgigas A uniquefeatureofallcoleoidcephalopods (squid,cuttlefishand Pop-up archivaltransmitting(PAT) tagshaverevealedagreat deal Despite theecologicandeconomicimportanceof also hasamajoreconomicpresence,currentlystandingasthe (Gilly etal.,2012;Bazzino2010;Stewart2012), 2 m, butduringthedayitmaintainsanaveragedepthof and GregMarshall D. gigas Dosidicus gigas D. gigas 2 D. gigas or otherommastrephids have onlyreddish-brown in situ hunts cooperativelyat observations at D. gigas, (Gilly 265 ) D. D.

The Journal of Experimental Biology simply reflects our muchsmallersamplesize. Different conditions patterns thanpreviouslyreported, itislikelythatthisdifference the head(notillustrated).Although weobservedconsiderablyfewer of thefins,prominentkeel on armsIIIandthedorsalsurfaceof lighting inROV observationsandinvolveddisplaysalong the edges to thosedescribedbyTrueblood (Trueblood, 2010)under artificial primary squidorsecondarysquid. Thesepatternswereverysimilar material Movie1). and thenjettedbackward,awayfromthecamera(supplementary squid andopenedtheirarmsasfarpossibleforseveralseconds primary squidapproached,theybothturnedtofacetheoncoming one case,apairofsecondarysquidwereinteractingandas the interactions thatdidnotappeartobedirectlyrelatedmating. In spermatophores beconfirmed. might alsorepresentmating,innocasecouldthetransfer of possibility thatitwasthehectocotylizedarm.Althoughthisbehavior appeared tobeoneofthemoreventralarms,consistentwith the (or )wasusedcouldnotbedetermined,butinsomeeventsit behavior involvingtheprimarysquid( observed severaltimes( some cases)totouchthebuccalareaofanotherindividualwere which asecondarysquidextendedonearm(orpossiblytentaclein were noarms-to-armscontactsofthetypedescribedabove.Eventsin observed severalpairsof‘secondary’ squidthatwereinteracting,there they werefemalebasedontheirextremelylarge size.Althoughwe We donotknowthesexofprimarysquids,butweassumethat did theactualarms-to-armscontactlastformorethanafewseconds. successful. A totaloffivesuchencounters occurred,andinnocase to bematingattempts,butwecannotascertainwhethertheywere camera-bearing (‘primary’)squid( squid withtheappearanceofspermatophoresbetweenarms During thisperiodmanyinteractionswithothersquidoccurred. extremely narrowrangefortheremainderofdeployment. a large groupofconspecificsandmaintaineditsdepthoveran before itquicklyascendedtothethermocline.Here,encountered whereas squid3remainedclosetoitsmaximumdepthfor~45 266 the squiduponreleaseatvelocitiesof0.5–1.0 All threeCrittercamdeploymentsshowedanimmediatedescentof coding, whereasothersquidmayrelymoreonspatialpatterning. displays appeartoprimarilyinvolvethetimedomainandtemporal gigas flickering canberapidlyandgloballyinterruptedor‘paused’. generally present,moresubtle‘flickering’.Bothflashingand intra-specific encounters(possiblyuniquetoommastrephids)anda squids), rapidandrhythmic‘flashing’ oftheentirebodyduring behavior wereobserved:staticpatterning(similartothatinloliginid during theseCrittercamdeployments.Threegeneraltypesof present paperdescribesdataonchromogenicbehaviorscollected the NationalGeographicCrittercam(Marshalletal.,2007).The RESEARCH ARTICLE from itsmaximumdepthtothethermoclineregion(50–60 off byconspecificsduringthedescent.Squid2slowlyascended except inthecaseofdeployment1,becauseCrittercamwastorn movements areevidentafterthesquidreachesitsmaximumdepth, characteristic of rather thanthe‘climb-and-glide’ patternoflocomotionthatis descents werecharacterizedbycontinuousdownwardmovement General behaviors of squid carrying aCrittercam squidcarrying of behaviors General RESULTS Eight staticchromogenicdisplayswereobservedineither the Splaying ofarmswasalsoobservedinseveralinstancesduring A numberoftheseinteractionsinvolved arms-to-armscontactof thus differs fromloligindsquidsinthatitschromogenic D. gigas i.2B,C),withoneobservationofthis Fig. (Gilly etal.,2012).Climb-and-glide i.2A).We taketheseinteractions Fig. i.2D).Exactlywhicharm Fig.

m

s − 1 Fg 1A).These (Fig.

min m), D. vertical movements oftheprimarysquidcan becomparedwith Because theCrittercamrecords depthandtemperature,thenatureof comparison ofthesedatasets. depths withartificialdaylight illuminationcomplicatedirect lighting comparedwithROV observationsmadeatmuch greater for daytimeCrittercamobservationsat<50 Crittercam attachedtotheHumboldtsquid Fig. Crittercam (circles)aresimilarovertherangeof±1 squid didnotshowhigh-velocityjetting,buttheseeventsareextremely rare. Vertical movements versus PAT byCrittercam recorded tags slowly todepthsof100 about 70 Crittercam deployment(1–3),thesquidrapidlydescendedtoadepthof distributions (meansin0.1 swimming andmorerapidverticalmovements.(C)Vertical velocity immediately afterrelease(atnight)andthenengageinclimb-and-glide terminated byattackfromconspecifics.(B)SquidcarryingPAT tagsalsodive about 50minthelateafternoon.Thedescentofsquid1wasprematurely climb-and-glide swimming.Bothsquidmovedtothethermoclinedepth at

.Comparisonofverticalmovements recordedbyPAT tagsand 1.

m afterrelease.Squids2and3thencontinuedtodescendmore Fraction of time Depth (m) 150 100 150 100 0.0001 50 50 0.001 0 0 0.01 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 0.1 0 B A C 0 1 11/08A 11/08B 1000 1000

m and160 11/08A –2 1

m Time afterdeployment(s)

s 3 2000 2000 − 1 Vertical velocity(ms bins) forsquidcarryingPAT tags(bars)and

m, respectively, andthencommenced –1 2 3000 3000 2 4000 4000 Dosidicus gigas. 0 3

m m depthundernatural

s 5000 5000 –1 − 1 ) . Crittercam-bearing 1 6000 6000 11/08B (A) Ineach 2

The Journal of Experimental Biology time. is notseriouslyhinderedforthevastmajorityofdeployment impairment offastjettingbytheCrittercam,itisclearthat squid based onourlimiteddataset.Althoughwecannotruleout some nature ofsuchfastmovementsthuscomplicatesthiscomparison exceedingly rare(Gillyetal.,2012).Therarityandidiosyncratic RESEARCH ARTICLE but delaysand timing ofthespreadactivity cannotberesolved within 1–2framesbyactivityon themantleandfins(notillustrated), secondary squidappearedtooriginate intheheadandwasfollowed The changefromlighttodark in acycleofflashingasobserved (Fig. change washighlycoordinated acrossthesurfaceofhead cases thedifference betweenpaleanddarkwaslarge, andthecolor 3B,C,seelaterresults),althoughinmost flashes variedwidely(Fig. flashes wereoccasionallyobserved.Theamplitudeofindividual 3B),butsingle generally involvedanepisodeofseveralcycles(Fig. 3A; supplementarymaterialMovie2).Thisdynamicdisplay to dark(red)andbackpalethatoccursovertheentirebody (Fig. ‘Flashing’ ischaracterizedbyarapidchangebetweenpale(white) of thetimeisspentatvelocitiesbetween logging device(forsimilaramountsoftime)showsthatover95% vertical-velocity distributionsforsquidcarryingeithertypeof 1B).Comparisonof as morerapiddescentsandascents(Fig. night) followedbyavarietyofclimb-and-glidemovementsaswell tags alsoshowasustaineddescentimmediatelyafterrelease(at those ofsquidcarryingmuchsmallerPAT tags.SquidcarryingPAT i.1C.Mostdescentsgreaterthan Fig. daytime (seeMaterialsandmethods)areindicatedbydatain direction) forthetwoCrittercam-bearingsquidreleasedduring the GulfofCaliforniareached±2 Maximum verticalvelocitiespreviouslyrecordedwithPAT tagsin seriously impairthesquidorgrosslyalteritsbehavior. versus aPAT tag.Thisstrongly suggeststhattheCrittercamdoesnot with noapparentdifference betweensquidcarryingaCrittercam All ascentsgreaterthan1.4 by PAT 11/08A, andtheywereassociatedwithonedeep,rapiddive. Dynamic chromatophore displays:flashing andflickering C AB A Virtually nofast-swimmingevents(>1 C n vrters ftebd scnaysudi i.3A). 3C) andovertherestofbody (secondarysquidinFig.

m

s − 1 were shownonlybyPAT 11/08B. −

m 1.2

s − m 1 , buttheseeventswere

s m − D 1

− s were displayedonly − 1 1

m in eithervertical

s − 1 and 1 m

s − 1 , lsig(i.3D). flashing (Fig. spatially coherentandtemporallysynchronizedasthatduring color duringflickeringisofmuchloweramplitudeandnot so 3BandsupplementarymaterialMovie3).Variation in skin Fig. has anoisywave-likeappearance(initialportionoftherecord in irregular chromatophoreactivityinneighboringpatchesofskin and the presenceandabsenceofconspecifics.Itischaracterized by that isgenerallyevidentwheneverflashingnotoccurring,both in by secondarysquidbutnottheprimarywasalsocommon. secondary squidwereoftenseenwithoutanyflashing,andflashing encounter initselfdidnotguaranteeflashing,becauseoneormore flashing occurredinconjunctionwithintraspecificencounters.An encounter occurredinthesecasesaswell.Thus,most,ifnotall, to thecamera,anditisreasonableassumethatanout-of-frame impossible todetectasecondarysquidthatwasnotextremelyclose of 8 secondary squidrangedfrom0.5 frame, thetimepreceding(orfollowing)flashingandimagingofa flashing. Forthesixepisodesthatoccurredwithoutanothersquidin was clearlyvisibleduringthetimeinwhichprimarysquid (91%) occurredinthepresenceofatleastonesecondarysquidthat flashing episodesindeployment2(67%)and403 during deployment2,and44eventsoccurredin3.Four head andfins(ourunpublisheddata). in thestudyarea2002alsoindicateadelayof<2 of flashingmadeat30 with theframe–capturerateofCrittercam(17 2.8±0.2 frequencies betweendeployments 2and3,3.5±0.3 rank-sum testshowedasignificant difference ( primary squidvariedbetween 2and4 instantaneous frequenciesduring anepisodeofflashingbythe time-course ofchromatophore activity. Theoverallmean of the cycleperiodbetweensuccessive palingminimainthecalculated Instantaneous frequencyofflashingwasmeasuredastheinverse of Flashing frequency Flashing ‘Flickering’ isaqualitativelydifferent type ofdynamicdisplay Six distinctflashingepisodesbytheprimarysquidwereobserved

s and21 Hz, respectively. Instantaneousfrequency often changed The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 s) occurredatdepthswithverydimlighting,makingit squid byextendingeitheranarmoratentacle(circled). squid makescontactwiththebuccalareaofasecondary by extendingoneofitsarms(circled).(D)Theprimary contact withthebuccalareaofanothersecondarysquid mating attempt.(B,C)Onesecondarysquidmakes combination ofthesefactorsisstronglyindicativea the primarysquidandasecondarysquid; accompanied byanarms-to-armsinteractionbetween squid. Thepresenceofspermatophoreswas (arrows) arevisiblebetweenthearmsofprimary 3. Crittercam deployment Fig.

.Potentialmatingattemptsobservedduring 2.

Hz duringdaylightbyadiverwithSCUBA

s to21

Hz (Fig. s. Two ofthesecases(gaps (A) Multiplespermatophores P

Hz). Observations =0.0048) inmean 4A). A Wilcoxon

frames between

Hz versus 267

The Journal of Experimental Biology (Fig. involved physicalcontactbetweentheprimaryandsecondarysquid during asingleflashingepisode,wassignificantlylessincasesthat as indicatedbythestandarddeviationofinstantaneousfrequency significant (Fig. contact wasmadewithanothersquid,butthisdifference wasnot frequency duringaflashingepisodewaslowerwhendirectphysical cycle inagivenepisode.Therewassuggestionthatmean maximum difference betweenmeanfrequencyandanindividual 268 flashing squidisindicatedinFig. secondary andprimarysquidin deployment3. during interactionsthroughfrequency changewasseenforboth of thetwosquidasillustrated inFig. thereby changingthepatternof thephaserelationshipfordarkening squid thenbegantoincreaseanddiverge fromthatofthesecondary, drifting inanoutofphase(Fig. frequency (2.2±0.1 Fig. of phaseiftheirfrequenciesarenotidentical.Inthecaseillustrated in two squidthataresimultaneouslyflashingtendstocomeinand out Because flashingisasinusoidal-likeglobaloscillation,thecolor of during anindividualflashingepisode,andFig. RESEARCH ARTICLE Timing of flashingbymultiplesquidduringinteractions Timing of (red trace)flashed ataregularfrequencyfor1.6 fairly constantinbothsquid(Fig. 1–7). Duringthistime,flashingfrequency(inversecycleperiod) was squid (2.8±0.2Hz;n C B Skin color (grayscale) Skin color (grayscale) A Another waytoacutelyalterthe phaserelationshipbetweentwo 100 200 100 200 5A theprimarysquid(blacktrace)displayedanaverage 4D). 0 0 0.0 .003 .20.47 0.42 0.35 0.30

4C). Changeoffrequencyduringaflashingepisode,

s

Hz; =9) forthefirst3 n =7) thatwaslowerthanofthesecondary

.601 .80.24 0.18 0.06 0.12 5C). Thecycleperiodoftheprimary

5B), withdarkeningofthetwosquid

6A. Inthiscase,asecondarysquid 1 s s oftherecord(primarycycles

5C. Modulationofflashing 0 5 s.d. D 100 200

s. A singleflashby 0

4B indicatesthe 5 s at theendofrecordinFig. example ofprolongeddarkening(byasecondarysquid)isevident mechanism forrapid,punctuatedcontrolofflashtiming,andanother during cycles6–9.Atcycle10,allthreesquidflashedinphase. at aconstantfrequencybutoutofphasewiththeothertwo squid secondary squid(onsetofcycle5).Thisthirdcontinuedflashing another secondarysquid(bluetrace)flashedinphasewiththeoriginal their extendeddarkperiod(centeredonthe2 secondary squid. aggression remainsunknown. of suchflashingvarieswithspecific interactionssuchasmatingor intra-specific signalingin strongly suggeststhatthisbehaviorisfundamentallyimportant to degree ofcontroloverthetiming,frequencyandphaseflashing effect betweentheindividual flashingrhythms.Theapparenthigh phase relationshipbetweenanimalsandproducesasyncopation-like then beganflashingat3 resumed atthepreviousfrequency(cycles6–10).Theprimarysquid darkening thatlastedforabout1 preceded byasignificant diminutionofflickering activityacrossthe About halfoftheflashingepisodes insquid3wereimmediately increase incycleperiod(redcirclesFig. of theprimarysquid’s darkperiod, andthisbehaviorledtoalarge displayed anextendeddarkperiod(cycle5)thatbeganinthemiddle the primarysquid(blacktraceat1 Temporal control of flickeringTemporal of control Extending adarkperiodinthiswaythusprovidesanother During thetimewhenbothprimaryandsecondarysquidwerein 1 s The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 0.53 0 5 s.d. 10×10 traces representskincolormeasuredin18 surface oftheprimarysquid’s head.Thelightred (C) Flashingishighlysynchronizedoverthedorsal before andduringanepisodeofflashing. skin coloraveragedovertheentireboxedarea most armsis~15 straight-line distancebetweenthebaseofouter- which skincolorwasquantified.Theprojected primary squidindicatestheapproximateareaover squid andaconspecific.Thewhiteboxonthe show out-of-phaseflashingbetweentheprimary Dosidicus gigas. Fig. with thedarkpeaksduringflashing. s.d. doesnotshowtheperiodicminimaassociated Spatial coherenceofflickeringasrevealedbythe the samesquidatadifferent timeinthevideoclip. (D) Flickeringwasquantifiedinananalogousway indicate ahighdegreeofspatialsynchronization. trace tendtooccurduringthepeakdarkperiodsand measurements. Minimainthestandarddeviation s.d. wascomputedfromthese18individual columns. Theheavyblacktraceisthemean.

s, whichwasmoreorlessinphasewiththe

.Flashingandflickeringbehaviourin 3. D. gigas.Howthestructure(i.e.syntax)

pixel boxesinanarrayof3rowsby6

5A. Thisbehaviorsuddenlyaltersthe

s. Thesecondarysquidthenalso

s) wasfollowedbyaprolonged

cm. (B)Time courseofchangein (A) Sequentialframesfromvideo

6B) beforeflashing

s markinFig. 6A)

The Journal of Experimental Biology RESEARCH ARTICLE decrease inmoving varianceoftheintensitysignal (lowertracein Fig. entire head,duringwhichtime theskinuniformlypaled(arrowsin with asecondarysquidwasmade(black barsvsbrownbars; episode showedsignificantlylessvariability (s.d.)ifdirectphysicalcontact difference wasnotsignificant.(D)Instantaneousfrequencyduring aflashing than thosethatdidnotinvolvedirectphysicalcontact(brownbars).This contact withasecondarysquid(blackbars)hadloweraveragefrequency (C) Inprimarysquid3,flashingepisodesthatcoincidedwithdirectphysical changes ininstantaneousfrequencyduringindividualflashingepisodes. squid 2(blackbars)andprimary3(whiteshowedsubstantial squid 2(blackbars)thanforprimary3(whitebars).(B)Both frequency forindividualflashingepisodeswassignificantlyhigherprimary Fig.

.FlashingfrequencyinDosidicusgigas. 4. 7A). This‘pausing’ behaviorcouldbedetected byalarge Number of events 10 12 10 12 10 10 12 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 16–. 1–.–. –0 –0.7–0.4 –1 –1.3 –1.6 . .50303 . .50505 . 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 D C B A ...... 4 3.8 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 2 22.22.42.62.83 Frequency change (Hz) Mean frequency (Hz) frequencyMean Mean frequency (Hz) frequencyMean s.d. 102050811141.7 1.4 1.1 0.8 0.5 0.2 .1 (A) Meanflashing P =0.0401). imitate features of thedynamicnatural-lightfield inthisintermediate Similarity inspectralcharacteristics suggeststhatflickeringmay amplitude ofsunlightvariations andflickeringwouldbecomparable. focused sunlightwouldbeasignificant signal,andatsomepoint,the thus benegligible.However, betweenthesurfaceand50 of theflickeringwasanalyzed, focused irradianceofsunlightwould this study(Dareckietal.,2011). Atdepths of discrete peaks,inthiscaseat2.2 covered alarge range,butpowerspectraweredominatedbystrong, then processedwithaFastFourierTransform. Flashingamplitude (0.5 and flashing(Fig. decreasing about10-foldin10 attenuates exponentiallywithdepthintheupperwatercolumn, respective Fourierspectra,focusingofdown-welledsunlight flickering asshownbythelarge difference inamplitudeofthe (Fig. those derivedfromflickeringatdepthoversimilarperiodsof time to episode(Fig. content wasevidentinsunlightfluctuationsthatvariedfromepisode flickering activityatdepth.Ineachcase,lowfrequencyspectral was carriedoutusingidenticalprocedurestothoseused for (supplementary materialMovie4).Analysisofsunlightfluctuations deployments 2and3beforetheanimalswerereleased natural sunlightfromthedorsalsurfaceofsquidindaytime The Crittercamrecordedreflectionsoffluctuationsdown-welled refraction bymovingripplesatthesurface(Dareckietal.,2011). column duetothefocusinganddefocusingofincidentsunlightafter on thebottomofapoolcausedbyfluctuationslightinwater frequencies. random ‘white’ noisethatwouldhaveaflatspectrumatall Flickering thusdoeshavesomespectralstructureandisnotsimply series ofweakerpeaksbetween1and4 Fig. series datafor~30 component frequenciesduringbothflickeringandflashing.Time- Spectral analysiswasundertakentoinvestigateunderlying another componentofchromogenicbehaviorin onset offlashing(Fig. several secondswasfollowedbyareturntoflickeringbeforethe association withflashing(notillustrated).Inonecase,apauseof similar pausesinflickeringsometimesoccurredwithnoapparent activity wasoftenobservedimmediatelybeforeaflashingepisode, from onlyafewframestoseveralseconds(Fig. Fig. preceded bywell-definedpauses. squid. Thistypeofsubduedflashingactivitydidnotappeartobe because wecanonlyobservesuchdetailsontheheadofprimary mode. We donotknowthespatialextentofthistypeactivity, coordinated toaconsiderableextentintomutedflashing-like the record.Theseobservationssuggestthatflickeringcanbe brief pausethatprecedesthestrongerflashingepisodeatendof synchronization, notillustrated)offlashingoccurringbeforethe (0.7–2.0 evident inFig. into aflashing-likebehavioroflowamplitude.Suchcoordinationis flashing, butinsomecasesflickeringappearedtosmoothlychange Spectral analysis of flickering andflashing of analysis Spectral Although down-welledsunlightismuchbrighterthanchromogenic To anobserver, flickeringresemblesthevariationsoflightseen Flickering doesnotshowthehighdegreeofsynchronization Hz cut-off frequency;Fig. 8C). Flickeringwasofmuchweakeramplitudeandshoweda 7A), andthedurationofsuchpausesvariedgreatly, ranging 9B). s) thatshowthetemporalcharacteristics(andspatial The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157

9A), andthesespectrawerequalitativelysimilarto 7D withfourcyclesoflow-amplitudeactivity 8B, toppanel)werefilteredwithahigh-passfilter

s ofuninterruptedflickering(Fig.

7C). Globalpausingofflickeringisthus

m underconditionssimilartothosein

8A andFig.

Hz, 2.6 Hz and2.75 8B, bottompanels)and

Hz (openredbars). ≥ 50

D. gigas. 7B). Althoughthis m, atwhichmost

8A, toppanel) Hz (bluebars,

m depth, 269

The Journal of Experimental Biology 270 of anycephalopodusingthisapproach.Threebasictypes California. Thisisthefirststudyofnaturalchromogenicbehavior an -bornevideopackageundernaturallightingintheGulfof In thispaper, wedescribebehaviorsofHumboldtsquidfilmedwith flickering behavior. upper watercolumnatdepthsoftensmetersmayinfluence These factorssuggestthatfluctuationsinexternallightcuesthe 3 whenflickeringcommencedshortlyafterthesquidwasreleased. illumination, flickeringwasnotobserved,unlikeindeployments2and deployment usingartificiallightthatprovidedconstantandstable region ofthewatercolumn.Furthermore,inonlyCrittercam RESEARCH ARTICLE DISCUSSION Cycle period (s) Skin color change (grayscale) Time between P and Cycle period Skin color change –40 –20

0.2 0.4 0.6 S

20 40 60 80 peak dark (s) (s) (grayscale) –0.2 0 0 –50 0.2 0.2 0.4 50 0 0 0 0 0 A B A C 0 B 0 – 12 1 ++ 1 1 1 1 34 –– 2 2 5 Time (s) Time (s) 54321 2 2 ++ 710 67 3 3 – 911 89 76 3 3 4 4 ++ 8 9 flickering andpausingaredemonstratedinthispaper. be understrongneuralcontrol.Directtransitionsbetweenflashing, and globallyturnedonoff, andthusbothtypesofbehaviormust the absenceofflashing.Bothflickeringandflashingcanbequickly subtle flickeringwithirregularwave-likecharacteristicsoccursin of squid(oranyothercephalopods)toourknowledge.Third,amore squid, butithasnotbeendescribedforspeciesinanyotherfamily oualaniensis 2–4 the entirebodyoscillatesbetweenpaleanddark,atfrequenciesof other speciesofsquid.Second,adynamicflashingoccursinwhich maintained forseveralsecondsaresimilartothoseseeninmany chromatophore displayarecommon.First,staticpatternsthat 10 5 5 Hz. Thistypeofdisplayalsooccursin 4 0 0.4 –0.4 4 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 corresponding s.d. omitting cycle5;thewidthofpink stripeindicatesthe line indicatesmeanfrequencyofthe firstsecondarysquid phase relationwiththeothersecondarysquid.Thedottedred in cycleperiodforthatsecondarysquidandalsoaltersthe extended darktimeincycle5correspondstoalargeincrease between minimumwhitevaluesasdescribedabove.The time ofmaximumdarkness,andcycleperiodwasmeasured records inpanelA.Timing ofthepointsisdeterminedby secondary squid(redandbluecircles)correspondingtothe time. (B)Cycleperiodsfortheprimary(blacksquares)and The othersecondarysquidcommencedflashingduringthis similar behaviorinoneofthesecondarysquid(red,cycle5). displays anextendeddarktime(1.5 and twosecondary(redbluetraces).Theprimarysquid flashing cycle. flashing squidbyextensionofthedarkperioda Fig. (our unpublisheddata),anotheroceanicommastrephid Fractional cycle lead

.Alterationofthephaserelationship between 6. or lag (P vs S) in phase(<20%cycleperiodlagorlead). Shaded boxesindicatetimeswhendarkcyclesare squid (fractionalcycleperiod,blacksquares). divided bytherelevantcycleperiodforprimary secondary squid(opencircles)andasthisvalue between peakdarknessoftheprimarysquidand was calculatedforeachcycle1–10asthetime for flashingofthetwosquiddepictedinA.Phase first 3 lines indicatemeanfrequenciescomputedforthe derivative ofcolorchange(notillustrated).Dotted cycle 1ofprimarysquid)asestimatedfromthe between palingminima(indicatedbyarrowheadsfor in A.Cycleperiodwasmeasuredasthetime in cycleperiodduringtheflashingepisodedepicted change crossedzero(notillustrated).(B)Changes interpolation ofthepointwhenderivativecolor by verticaldashedlines.Timing wasestimatedby maximal darknessoftheprimarysquidasindicated Numbered cycles1–11 refertothe timingof than themeanvalue(positivevalues)areshaded. to facilitatecomparison.Times withskincolordarker values fortheentiretimecoursearesuperimposed (red trace)squidrelativetotheindividualmean color oftheprimary(blacktrace)andasecondary between twoflashingsquid Fig.

.Alterationsinthephaserelationship 5. (A) Flashingrecordsforaprimary(blacktrace) s. (C)Time courseofthephaserelationship − 2.5 . (A)Changeinskin s) followedbya Sthenoteuthis

The Journal of Experimental Biology RESEARCH ARTICLE – from1 been describedasbeingofmuch longerdurationthanreportedhere observed, butputativearms-to-arms encountersin Dawe, 2013).Matingofommastrephidsinthewildhasrarely been pacificus laboratory observationsoftwootherommastrephids, attempts. Matingencountersofthissorthavebeenreported in squid. Theseobservationssuggestbrief,arms-to-armsmating spermatophores inthemidstofsplayedarmsprimary deployments 2and3basedontheunambiguousappearance of Putative matingattemptswerecapturedbyCrittercamduring both observed byCrittercam. Morethanonetypeof matingbehaviorhas situation mayleadtoadifferent typeofmatingbehaviorthan that from thedeckofavesselatnight usingartificialillumination.This et al.,2006).Bothofthesereports werebasedonobservationsmade column). relevant environmentalcues(e.g.lightfluctuationsinthe water spectral analysestorelatespecificbehaviors(e.g.flickering) intra-specific communication(syntax,etc.).Thisfeaturealsopermits as one-dimensionaltimeseries,amajorsimplificationinanalysisof Thus, thesechromogenicbehaviorsof flashing andpausingremovesthespatialaspectsofthesedisplays. eliminates thecolordimension.Second,dynamicglobalcontrolof behavioral analysis.First,thesingle-colorchromatophoresystem Mating behavior in Skin color (grayscale) Two majorfeaturesofthesechromatophoredisplaysfacilitate 160 120 160 120 160 120 80 40 80 40 80 40 160 120 80 40 0 0 min (Nigmatullinetal.,2001) to morethan10 0 A (Sakurai etal.,2003)and C B 0 D 0.5 2 1.0 2 2 Dosidicus gigas Dosidicus 1.5 Time (s) 4 2.0 Time (s) 4 4 6 2.5 Time (s) 3.0 Illex illecbrosus Time (s) D. gigas 8 3.5 6 6 0 5 10 15 20 25 10 can berepresented Variance D. gigas 8 8 (O’Dor and 12

Todarodes min (Gilly 0 5 10 15 20 25 have 14 Variance 10 16 transmitted between squidremainunknown. may modulatethebehaviour andwhatsortofinformationis view. Whatcircumstancestriggerflashing,whatbehavioral context behavior thatissimplytriggered wheneveranothersquidcomesinto behavior. Butflashingisclearlynotafixed-action-patterntypeof intraspecific signalingandthat visual contactisakeydriverofthis These observationsstrongly suggest thatflashingrepresents many flashingexchangesdidnotinvolveanyphysicalcontact. but flashingoftenbeganbeforephysicalcontactwasmade, and primary andsecondarysquidwasalwaysassociatedwithflashing, absence ofaconspecific.Directphysicalcontactbetween the Flashing bytheprimarysquidwasessentiallyneverobserved in the wild, sothetimingbetweentheseeventsremainsunclear. 2013). Naturalmatingandegg-layinghavenotbeenobserved in the pelagic eggmasswiththesurroundingseawater(O’DorandDawe, depth mightbeguaranteedbysimplyequilibratingthedensity of the maximum densitygradient),thenembryonicdevelopmentat this thermocline (whichisgenerallyequivalenttothepycnoclineor by Crittercam.Ifmatingandegg-layingbothtookplacenearthe 2008), similartothedepthatwhichmatingattemptswereobserved suspended atthethermoclineinGulfofCalifornia(Staafetal., not unexpected.Theonlyeggmassreportedthusfarfor 2013). been observedinIllexillecebrosus Flashing and intra-specific interactions and intra-specific Flashing 0 5 10 15 20 25 Mating nearthethermoclinedepthasobservedwithCrittercamis Variance 0 5 10 15 20 25

Variance The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 (A) Flickering(redtrace)duringthefirst8.5 Fig. commences. by apausebeforeanepisodeofstrongflashing into alow-amplitudeformofflashing,inthiscasefollowed progression toflashing.(D)Flickeringcanbecoordinated can beinterruptedbyapausewithoutanimmediate pause inflickeringbeforeaflashingepisode.(C)Flickering trace) offlickering.(B)Anexampleanunusuallylong corresponds toalargereductionintherollings.d.(blue The pauseinflickering(timebetweenarrowheads) paling immediatelybeforetheonsetofaflashingepisode. is followedbyreducedleveloffluctuationandoverall

.Pausingofflickeringbeforeaflashingepisode. 7. in captivity(O’DorandDawe,

s oftherecord D. gigas 271 was

The Journal of Experimental Biology system. Unfortunately, weknowlittleoftheseprocessesin the centralnervoussystemandflicker-fusion frequency ofthevisual chromatophore neuromuscularsystem,synchronizationcapability of that involves(ataminimum)thephysiologicallimitsof the 272 was presenttoimage satisfactorily, anditwould appeartorepresent Flickering wasalwaysobserved wheneveradequatenaturallight RESEARCH ARTICLE parameters anddepthwouldprovideinsightintothisquestion. deployments inlocationswithdifferent relationshipsbetweenthese on thisbehaviorcouldnotbeascertained.FutureCrittercam effects ofabioticfactorssuchas temperatureandoxygenconcentration behavior couldberesolvedundernaturallightingconditions, the Unfortunately, becauseofthelimitedrangedepthatwhichflashing behavior isafundamentalformofintraspecificsignalingin over flashingdynamicsisconsistentwiththeproposalthatthis of thesechangesareunclear. Regardless, suchahighdegreeofcontrol adjusted byanindividualsquid,butthemotivationforandeffects high degree.Flashingamplitude,frequencyandphasecanallbe component involvingvertebrate predators. the questionwouldalsohave asignificantco-evolutionary Additionally, ifflashingservesassomesortofpredatordeterrence, Flickering andcrypsis ‘Power’ flash Flash grayscale Flicker grayscale What setstherangeof2–4 Several observationsindicatethatflashingcanbecontrolledtoa –100 –20 –10 250 200 150 100 250 200 150 100 –50 100 100 150 200 250 50 10 20 50 50 50 0 0 0 0 0 0 0 0 0 0 B A C 5 5 5 5 2

10 10 Hz forflashingisacomplexquestion 10 10 Frequency (Hz) 4 15 15 15 15 Time (s) 20 20 20 20 6 D. gigas D. gigas. 25 25 25 25 . coordination of individualchromatophores,but flashinghasan characteristics suchasspeed ofpatterngenerationandstrict 1994; Hanlonetal.,1999;Trueblood, 2010).Thesebehaviorsshare system asthestaticpatternsseen inloliginidspecies(Hanlonetal., Flashing inD.gigas various anglesanddepthsremainstobeestablished. et al.,2012).Howflickeringwouldbeperceivedbypredators from shallow, better-illuminated depths,regardlessoftimeday(Gilly value particularlysincethissquidoftenmakesexcursions into mimics downwelledsunlightstrikingitsbodymightbeof some patterning thatresemblesobjects,butdynamic that oceanic environment, benthic features(HanlonandMessenger, 1996).Livinginan associatedwithcoastalloliginidsquidthatmimic static this oceanicsquidandprovidesafunctionanalogousto the propose thatchromogenicflickeringprovidesdynamiccrypsis for chromatophore musculatureorneuronalactivationpathway. We composition suggeststhatitisnotsimplyrandomnoiseinthe muscle toneinaconventionalmotorsystem,theunderlyingspectral flashing. Althoughflickeringmightbeconsideredanalogousto a basallevelofchromatophoreactivitythatoccursintheabsence gigas Chromogenic behaviors and control pathways andcontrol behaviors in Chromogenic 8 30 30 30 30 versus othercephalopods 0 0.1 0.2 0.3 0.4 0.5

‘Power’ flicker The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 centered around2.6 spectrum forflashing(bluebars)featuresstrongpeaks (C) AmplitudeoftherealpartFast-Fourierpower were processedinthesamemannerasforflickering. fluctuations (bottom).(B)Dataforcontinuousflashing with ahigh-passfilter0.5 measured overa20×20pixelsquare(top)wasprocessed (A) Thirtysecondsofcontinuousflickeringactivity Fig. much weakerpeaksinthe1–4 frequency limitissetbytheNyquist(8.5 because ofthehigh-passfiltering,andupper The 0–0.5 the videosamplingrateused.

.Spectralanalysisofflickeringandflashing. 8. is likelytoberootedinthesame neural-control D. gigas

Hz rangehasbeenomittedfromthesedata might havelittleneedforfeature

Hz. Flickeringischaracterizedby

Hz range(openredbars).

Hz cut-off toreveal Dosidicus

Hz) for

The Journal of Experimental Biology RESEARCH ARTICLE D. gigas or byparalleltracts. synchronous motorinputscould betransmittedbythesameaxons higher centersofthebrain(Boycott, 1961).Asynchronousand lobes wheretherelevantmotor neuronsaresubjecttocontrolfrom must occurduringflashing)would takeplaceinthechromatophore switching fromasynchronoustosynchronousmotoractivity (as patches ofskin(i.e.severalmotorunits).Ifso,itwouldseem that activity indescendingmotoraxonsthatprojecttorelatively small control couldbeanimportantfactor. nature offlickeringin chromatophores arenotwellunderstood,buttheirregularwave-like and pathwaysunderlyingmyogeniccontrolofcephalopod belong, mustbeoperative(Packard,1995b).Relevantmechanisms radial musclefibersthemselvesandtothenetworkswhich they motor control,andthussomemyogenicinherentto the denervated conditionsdoesnotdependonordinarydescending () opalescence (Packard, 1992)andindenervated wave-like chromatophoreactivityindenervated phenomena mayrepresentrelatedprocesses. amplitude, irregularflickeringwedescribein both aspectsminiatureoscillationsthusshowsimilaritytothelow- occur inbackgroundareasofminimalchromatophoreactivity. In 2–7 lessoniana (and smallgroups)havebeendescribedintheloliginid, cycles ofpartialexpansion/retractioninindividualchromatophores analogous processmayoccurinotherspecies.Rapidoscillatory described inothercephalopodsasadistinctbehavior, butan be moresignificantforloliginidspecies. primary importancefor patterning. Signalinginthetime-domainthusappearstobeof accelerated andhighlysynchronizedtimescalealackofspatial ‘Power’ Pausing isanotherimportantfeature ofchromogenicbehaviorin Alternatively, flickeringcouldrepresentasynchronousoscillatory Flickering alsosharessomecharacteristicswithspontaneous The dynamicdisplaywedefineas‘flickering’ hasnotbeen 120 160 Hz areprominentinthefeatureareasofpatterns,buttheyalso 40 80 10 20 30 40 10 20 30 40 50 0 0 0 0 0 0 AB that operatesin a synchronousmode,becauseboth flashing (Suzuki etal.,2011). These‘miniatureoscillations’ of 2 2 2 (Packard, 1995a).Suchactivityinchronically D. gigas D. gigas,whereasthespatialdomainmay 4 4 4 suggests thatperipheralmyogenic 6 6 6 Loligo vulgaris 8 8 8 Frequency (Hz) D. gigas,andboth Octopus vulgaris and 0.4 0.8 1.2 1.6 0.2 0.4 0.6 10 Sepiotuethis 0 0 2 4 6 8 0 Doryteuthis 0 0 0 2 2 2 comparison ofvertical movementsforsquidbearing PAT tags versus following releaseofthesquidwere re-analyzedinthisstudytoprovidea described previously(Gillyetal., 2012), butthefirst6000 archival records.Methodological detailsanddatafromthesetagswere Physical recoveryofthePAT tagsafter2 deployments, andthesampling rate (1 were deployedinthesamelocation andtimeofyearastheCrittercam 112.32°W). ThesePAT tags[11/08A (PTT 64004)and11/08B (PTT 83048)] California coastoftheGuaymasBasin,Gulf(27.53°N, research vesselBIP XIIduringthenightof8Nov. 2008off theBaja on twolarge Humboldtsquid (77and79 PAT tags(MK10,Wildlife Computers,Redmond,WA, USA)weredeployed mechanisms arelikelytobefundamentallysimilar. domain mayvarywidelyacrosstaxa,buttheunderlyingcontrol The relativeimportanceofsignalinginthetemporalversusspatial face ofremarkablestructuralconservationremainstobedetermined. functions ofthisuniquechromatophoresystemhavediverged inthe evolutionary historyofthisgroup.To whatdegreespecialized neural mechanismsofchromatophorecontrolappearedearlyinthe (Hanlon andMessenger, 1996).Itisthereforelikelythatthebasic , octopus)includebothstaticanddynamicpatterning pathways forommastrephidsquids. concerning eitherexcitatoryorinhibitorycontrolmechanisms unknown peripheralneuralplexus.Currently, therearenodata the stellateganglion(Gonzalez-Bellidoetal.,2014)orsome this pathwayinvolvesaxonsoriginatinginthechromatophorelobes, inhibitory serotonergic controlintheperiphery. Itisunclearwhether (Messenger etal.,1997;Messenger, 2001),stronglysuggests potent inhibitor)inperipheralaxonalprocessesloliginidsquids cephalopod chromatophoresystem,thepresenceofserotonin(a inhibitory motorinnervationhaseverbeenconfirmedinthe (or both)cannotbespecifiedatthistime.Althoughnodirect control, butwhetherthisinhibitionoccurscentrallyorperipherally highly timedbehaviorundoubtedlyinvolvesactiveinhibitory and flickeringcanbegloballyhaltedveryrapidly. Controlofthis Pop-up (PAT) archivaltransmitting tags MATERIALS ANDMETHODS Chromogenic displaysinallcoleoidcephalopods(squid, 4 4 4 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 6 6 6 8 8 8 for 7.1 of thewatercolumn.Datawerecollected sunlight withthesquidinupper1–2 of spectraforfluctuationsreflected head oftheprimarysquid. chromogenic flickeringviewedonthe of downwelledsunlightversus Fig. Average depthsforsquidwere43.5 Data werecollectedfor5.8 fluctuations areofverylowamplitude. flickering atdepthwheresunlight-related (bottom). (B)Analogousdataforperiodsof (top), 47.2

.Spectralanalysisoffluctuations 9. s (top),16.5

Hz) wasidenticalinallcases.

week deploymentsyieldedfull m (middle)and50.1

cm mantlelength)fromthe s (middle)and9.1

s ineachcase. s ofrecordings (A) Examples

m (bottom).

m

s 273

m

The Journal of Experimental Biology 2010). cannibalism, behaviorsthatarecommonfor light andthatilluminationofthissortatnightcanleadtoaggression and without LEDilluminationstronglysuggeststhatHumboldtsquidcansee red while theCrittercamwasfloatingtosurface.Theabsenceofsuchattacks during thisdeploymentshowsnumerousattacksmadebyothersquid,even the camera-bearingsquidshortlyafteritwasreleased(seeFig. terminated whenseveralothersquid( wavelength) LEDsforillumination.Thismissionwasprematurely charter vessel using rodandreelwithweightedluminescentjigs(51 (5 Sept.2009)wascarriedoutaftersunsetandusedred(700 velocity cannotbedetermined. velocity andorientationofthesquid,horizontalcomponentoraxial sensor datafromtheCrittercamcanresolveverticalcomponentof recovered inthefieldtoallowretrievalofrecordeddata.Although sea surfaceandtransmitsaVHFsignalbywhichitcanbelocated platform onthecamera-bearinganimalataprogrammedtime,floatsto Crittercam andinitssyntactic-foamcradledetachesfromamounting as thoseinthisstudy, is0.02 without artificiallighting,andshutterspeedunderdimlightconditionssuch (720×480 pixels)black-and-whitevideoat17 120 surface temperature,28.5°C;thermoclinedepth,50–60 depth was~16.5°C.CorrespondingdatarecordedbyCrittercamwere: camera wasrecoveredwithin3 end shortlybeforesunsetat18:30 late afternoonusingnoartificiallightingwiththemissionprogrammedto (Gilly etal.,2012). vigorously beforebeingreleased.Additionaldetailsaregivenelsewhere beneath theocean’s surfacebythedivertoconfirmitsabilityjet jig wascarefullyremovedfromthearms,andsquidgentlyheld forward view, i.e.towardsthearms.OnceCrittercamwassecured, all cases,theCrittercamwasorientedparalleltomantleaxiswitha with theattachedCrittercamwasthensecuredtomountingplatform.In at theanteriorendinordertostabilizesleeveassembly. A foamcradle ties werealsopassedthroughthesleeveanddorsaledgeofmantle securely aroundthemantleusingcabletiesatposteriorend.Two cable plastic mounting-platformwasattached,slidoverthefinsandcinched a diverinthewater. A stretchable,syntheticclothsleeve,towhichathin manipulate thesquidusingjigthatwasstillattachedtoarms,andby member positionedontheswim-stepofvessel,whowasableto For alldeployments,thesquidwasheldjustundersurfacebyateam 274 was analyzedforintensity changesandcorrectedformovements ofthesquid’s video, andaselectable arrayofsquareareasontheimaged surfaceofthehead surface oftheheadprimary squid isalwaysvisibleintheCrittercam permit automatedanalysisofdynamic chromogenicbehaviors.Thedorsal squid. the primarysquidandconspecifics thatwereencounteredaresecondary interaction. Inthispaper, thesquidcarryingCrittercam isreferredtoas squid, orthepresenceofvisiblespermatophoresovercourse each taken ofthepresenceothersquid,anydirectphysicalcontactwithanother and documented,alongwithanydistinctphysicalpostures.Notewas also USA). Startandendtimesofallvisiblechromogenicbehaviorswerelogged Matlab ImageProcessingToolbox (v8.0TheMathWorks Inc.,Natick,MA, Cambridge, MA,USA),andtheresultingimageswereanalyzedusing the Videos weresplitintoimage stacksusingVirtualdub (v1.9.11, Avery Lee, accelerometer dataatarateof1 This instrumentpackagerecordsdepthandtemperatureplustri-axial 23°C, anddepthofthethermoclinewas40–50 Crittercam. Average sea-surfacetemperaturerecordedbythePAT tagswas RESEARCH ARTICLE Video analysis Video Crittercam Large specimensof Deployments 2(6Sept.2009)and3(7werecarriedoutin Matlab-based softwareandagraphical user-interface weredevelopedto m depth,~17.5°C. Sandman Dosidicus gigas (deployments 1–3:Sept.2009;27.54°N,112.3°W). s. Thepackagestoresalldataonboard.

miles ofthedeploymentsite.Deployment1

h localtime.Inbothcases,thefloating

Hz andcapturesstandard-resolution D. gigas)torethecamerapackageoff (>80 cm mantlelength)werecaught D. gigas

Hz. Itcanbeusedwithor

m. Temperature at120

cm length)aboardthe

(Ibáñez andKeyl, m; temperatureat

1). Video

nm

m analyzed (>50 and thatthesunlightsignalisnegligibleatdepthswheremostflickeringwas assume thatthechromatophoresignalisnegligibleundertheseconditions 10–100 timesmoreintensethanrelevantchromatophoreactivity, andwe chromatophore activity. Sunlightfluctuationsattheseshallowdepthsare data wereanalyzedwiththeidenticalproceduresasthoseusedfor at least100individualchromatophores. in thesamewayfordorsalmantle(1480±174 was heldbeneaththeseasurface(upto2 squid’s headwererecordedwithCrittercamduringthetimewhensquid Fluctuations ofdown-welledsunlightonthedorsalsurfaceprimary the spatialscalespecified,generally10×10pixels(~0.1 during eachframeandplottedasatime-seriesofchromatophoreactivityover value (0–255,with0beingwhite)wassummedforallthepixelsineacharea unchanging anatomicalfeaturethatservedasalandmark.Thetotalgrayscale head (yawingtotheleftandrightof<±30 http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.114157/-/DC1 Supplementary materialavailableonlineat National GeographicTelevision (DangerousEncounterswithBradyBarr). the CensusofMarineLifeproject,Tagging ofPacificPelagics(TOPP); and Society (grantnumber8458-08);theGordonandBettyMooreFoundationthrough (grant numbersOCE0850839andIOS1420693);theNationalGeographic N000140911054 totheUniversityofTexas), theUSNationalScienceFoundation This workwassupportedtheUSOffice ofNavalResearch(grantnumber edits tothemanuscript. technical andscientificoversightfortheCrittercamdeploymentsprovided undergraduate honorsthesisandprovidededitstothemanuscript.G.M. a preliminaryanalysisofvideoandsensordatafromCrittercaminan Crittercam deploymentsinthefieldandprovidedmanuscriptedits.L.B.carriedout time-course dataandassistedwithmanuscriptpreparation.K.A.carriedout Crittercam deploymentsinthefield,analyzedPAT tagdataandchromatophore H.R. analyzedallvideodataandpreparedthemanuscript.W.G. carriedoutthe The authorsdeclarenocompetingorfinancialinterests. ‘Devils oftheDeep’. Television foraccesstorawvideodatacollectedinconjunctionwithproductionof manuscript. We alsothank BobCranston,SueHoughtonandNationalGeographic Andrew Packardformanythoughtfullythornydiscussionsandcommentsonthe Stramski andMirekDareckiforvaluablediscussionunpublisheddata, Patrick DanielandAshleyBoothforaidinanalyzingPAT tagdata,Dariusz assistance inthefield,RussellWilliamsforprogrammingandspectralanalysis, We thankRonSteel,Ted Uyeno,BradyBarr, SimonBoyceandJoelHollanderfor 1332±155 0.5×0.05 using ahigh-definitionvideocameraontripod.Patchesofskin of afreshlysacrificedDosidicusgigas of head)or20×20pixelsarectangulararraysuchsquares. 10×10 pixelsontheimagedheadwouldcorrespondto~0.1 oct,B. Boycott, Ramos- W. F. Gilly, and and K.J. C.A. Benoit-Bird, Salinas-Zavala, U., Markaida, W. F., Gilly, G., Bazzino, ulc,T .adHrig,G.A. Horridge, and T. H. Bullock, Oceanic lightfluctuations References Supplementary material Funding Author contributions Competing interests Acknowledgements officinalis jumbo squid . Prog.Oceanogr. the jumbosquid( J. Castillejos, Company. System ofInvertebrates Chromatophore densitywasmeasuredonthedorsalsurfaceofhead cm insizewereanalyzed,andchromatophoredensitywas . Proc.R.Soc.B cm Dosidicus gigas The JournalofExperimentalBiology(2015)doi:10.1242/jeb.114157 − (1961). Thefunctionalorganizationofthe brainofthecuttlefish

2 m). (2010). Horizontalmovements,vertical-habitat utilizationanddietof (mean ±s.d.; Dosidicus gigas Vol. II,pp.1435. SanFrancisco,CA:W.H. Freeman and , 86, 59-71. 153 . Mar. Ecol.Prog.Ser. , 503-534. n (2012). Coordinatednocturnalbehavior of foraging =7). Thisvaluewassimilartothatobtained ) inthePacificOceanoff BajaCaliforniaSur, (1965). (50 Structure andFunctionintheNervous cm mantlelength)underseawater

m depth)priortorelease.These

deg) bytrackingbasedonan 455 , 211-228.

cm

cm − 2 2 ; on imagedsurface n

cm =6). Anareaof 2 and contain

The Journal of Experimental Biology RESEARCH ARTICLE uh .L,Rbsn .H n adel R.L. Caldwell, and B.H. Robison, S.L., Bush, akia .adSs-ihzk,O. Sosa-Nishizaki, and U. Markaida, aek,M,Srmk,D n oosi M. Sokolski, and D. Stramski, M., Darecki, FAO (FoodandAgricultureOrganization) Davis R.W., Jaquet,N.,Gendron,D.,Markaida,U.,Bazzino,G.andGilly, W. Field, J.C.,Elliger, C.,Baltz,K.,Gillespie,G.E.,Gilly, W. F., Ruiz-Cooley, R.I., il,W . edeg .D,Boh .A . twr,J . asal . Abernathy, G., Marshall, J.S., Stewart, J.A.T., Booth, L.D., Zeidberg, W. F., Gilly, il,W . lie,C . aia,C . aail-op . azn,G and G. Bazzino, S., Camarilla-Coop, C.A., Salinas, C.A., Elliger, W. F., Gilly, E. Florey, ozlzBlio .T,Wril .J,Brsh .C,Umr .M n aln R. Hanlon, and K.M. Ulmer, K.C., Buresch, T. J., Wardill, P. T., Gonzalez-Bellido, aln .T,Sae .J n ae,W. H. Sauer, and M.J. Smale, R.T., J.B. Hanlon, Messenger, and R.T. Hanlon, aln .T,Mxel .R,Sahr . ow .R n ol,K.L. Boyle, and E.R. Loew, N., Shashar, M.R., Maxwell, R.T., Hanlon, ut .C n ebl B.A. Seibel, and J.C. Hunt, uoea . oaa .adMr,K. Mori, and Y. Koyama, T., Kubodera, F. Keyl, and C.M. Ibáñez, danae Octopoteuthis deletron locomotor, chromatic,postural,andbioluminescentbehaviorsofthedeep-seasquid Mexico. J.Mar. Biol.Assoc.UK Dosidicus gigas Radiometer System. light fluctuationsinducedbyseasurfacewaveswithandUnderwaterPorcupine 291-302. species, thejumbosquid,inGulfofCalifornia,Mexico. (2007). Divingbehaviorofspermwhalesinrelationtoamajorprey Zool. Current System. ecology andmovementpatternsofjumbosquid( Pearse, D.,Stewart,J.S.,Matsubu,W. andWalker, W. A. Species in2012 . el .E n el L.E. Dosidicus gigas andBell, L.E. Bell, K., basin, GulfofCalifornia,Mexico. M. Beman, Exp. Biol. peripheral ganglioncontrol. T. 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