974 Received 22July 2013; Accepted12November2013 *Author ([email protected]) for correspondence MI 49008,USA. BiologicalSciences, Michigan Western University, of Department Kalamazoo, protection (Banaszak,2003).Behaviorally, someanimals–especially 2003) aswellscreeningpigmentsorreflectivesurfacesthat offer repair mechanismstodealwithdamagecausedbyUVR(Buma et al., periods (BlumthalerandWebb, 2003).Manyanimalshaveevolved available atthesurfaceofearth,nowandoverevolutionary referred tohereasUVR)islessattenuated,andthereforethe most nm; by atmosphericozone.Nearultravioletradiation(UV-A; 320–400 2010; SinhaandHäder, 2002),althoughthey areheavilyattenuated cause significantdamagetonucleicacidsandproteins(Rastogi et al., nm]areofparticularbiologicalinterestbecausetheycan 280–320 wavelengths. Theshorterwavelengths[ultravioletB(UV-B); the humaneye,solarspectrumcontainsbothlongerandshorter respond toitinadaptiveways.Inadditionvisiblelightdetected by Virtually allanimalshaveevolvedneuralcircuitstodetectlightand Ultraviolet radiation KEY WORDS:,,Leech,Light,Escape, visible light. UVR andisabletodiscriminatebehaviorallybetween head andtail.Theseresultsdemonstratethattheleechcandetect behavioral responses,theS-cell,wasactivatedbyUVR,onboth UVR, andahigher-orderneuronassociatedwithshorteningrapid individual leechphotoreceptorsalsoshowedstrongresponsesto the cephaliceyesrespondedvigorouslytoUVR.Additionally, UV lighttothetail.Electrophysiologicalnerverecordingsshowedthat shortened awayfromUVlighttothehead,andextended stimuli. Theresponsesdependeduponthestimulussite:leeches responses infedandunfedleecheswerecomparableforUVR responses tovisiblelight(respondingin~8%ofthetrials).The responding in100%oftrialscomparedwithmodestnegative leeches alsoexhibitedavigorousnegativephototaxistoUVR; only aslightnegativephototaxistogreenandwhitelight.Individual phototaxis toUVR,buthadnobehavioralresponseblueorredand Groups ofunfedjuvenileleechesexhibitedarobustnegative nm). leeches coulddetectandspecificallyavoidnearUVR(395–405 I usedhighintensitylightemittingdiodestotestthehypothesisthat order toavoidpotentialultravioletradiation(UVR)-induceddamage. do notappeartobeasnegativelyphototacticonemightexpectin been showntorespondvisiblelight,leechesinthegenus exposed tolessscreenedlight.Whereastheleechvisualsystemhas during daylightandalsoleavethewaterwheretheymightbe Medicinal leechesareaquaticpredatorsthatinhabitsurfacewaters John Jellies* an aquaticannelid:themedicinalleech Detection andselectiveavoidanceofnearultravioletradiationby RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,974-985doi:10.1242/jeb.094243 INTRODUCTION ABSTRACT Hirudo are phaosomalprimary receptorneuronsthatdepolarize inresponse responses tovisiblelight(Kretz etal.,1976).Leechphotoreceptors sensillar photoreceptorsaresimilar tothecephaliconesintheir photoreceptor cells(Friesen,1981; DerosaandFriesen,1981).The detection ofwaterwaveand othervibrations,aswell et al.,1976).Thesesegmental sensillacontainhaircellsusedfor sensilla distributeddorsaltoventral alongthecentralannulus(Kretz cephalic eyes,eachmidbodysegmenthassevenpairsof small join fourcephalicnerves(Kretzetal.,1976).Inadditionto these photoreceptors, andtheaxonsoftheseareprojectedinbundles that Each ofthesecephaliceyescontainsapproximately50individual 1). eyecups alongtheanteriormargin oftheanteriorsucker(Fig. surface (Kretzetal.,1976).Therearefivepairsofpigmented eyes ontheheadandmultiplesimple,‘sensillar’ eyesalongthebody no reportofleechesdetectingorrespondingtoUVR. Peterson, 1984b;1985a;1985b),therehas been photoreceptors withinthecentralnervoussystem(Laverack, 1969; well asstudiescharacterizingsomeoftheinterneuronaltargets of wavelengths (Kretzetal.,1976;Laverack,1969;Walz, 1982),as on thevisualsystemofleechesshowingsensitivitytovisible Harley etal.,2013).Althoughtherehavebeenanumberofstudies McVean, 1993;DickinsonandLent,1984;Harleyetal.,2011; water wavesthatmightbeproducedbypotentialprey(Carltonand been showntoorienttowardmovingbarsoflightassociatedwith that hungryleechesaresomewhatpositivelyphototactic,andhave phototaxis tovisiblelightisfurtherreducedbyfeedingstatus,such with artificialsourcesofvisiblelight(Mann,1962).Thisnegative genus vulnerable toUVR-induceddamage.Despitethis,leechesinthe (juvenile, hatchlingleeches)wouldseemtobeparticularly crawl somedistanceinordertolocatethewater. Thisstageoflife Indeed, never-fed juvenileshatchfromcocoonsonlandandmust and leavethewatertodepositcocoonsonland(Sawyer, 1986). Additionally, medicinalleechesofthegenus surface waters(Sawyer, 1986)whereUVRcanbehigh. the environment. and Morris,2003)mightthereforebecorrelatedwithUVRin oxygen levelsandturbidityaffect aquaticUVRattenuation(Osburn and Roy, 1993).Furthermore,otherfactors,suchasdissolved Johnson, 2003;Reizopoulouetal.,2000;Tank etal.,2003;Vincent vertebrates andinvertebrates(Boeingetal.,2004;Leech 2003). Thus,UVRisapotentiallysignificantfactorforaquatic amounts ofUVRcanpenetrateintothewatercolumn(Hargreaves, until recently. Ithasbecome increasinglyclearthatsignificant al., 2001)andnavigation(Tovée, 1995)behaviors. that isusefulinmateselection(Landetal.,2007),foraging(Kevan neuronal receptorsandcircuitsthatuseUVRtogatherinformation those thatalsohaveeffective UVRscreening–havealsoevolved Medicinal leecheshaveaspecializedvisualsystemwithcephalic Aquatic predatorssuchasmedicinalleechescanbeactivein Aquatic systemshavereceivedlessattentionregardingUVR, Hirudo are onlymodestlynegativelyphototacticwhentested Hirudo are amphibious

The Journal of Experimental wavelength (P response (Fig. were comparedacrosstime,itwasclearthatUVRevokedarobust evoked achangeinmovement.Whentheresultsofallwavelengths was stationaryorswimmingcrawling–exposureto UVR regardless ofwhethertheleechwasenteringorexitingquadrant, the fractionofindividualsthatleftquadrant.Itappeared – quadrant wasclearofleeches.Theavoidancefraction( smostofthe leeches beganmovingwithinafewseconds,andby15 2A).WhenUVRwasshoneontoaquadrant, reaction toUVR(Fig. The clearestandmostrobustresponsewasanegativephototactic capture allresponses. swouldeasily few seconds,sothatatotalexposuretimeof60 responses, iftheyweregoingtooccuratall,wouldbeginwithina of atransparentdish.Preliminaryexperimentsshowedthat different lightemittingdiodes(LED)positionedoveronequadrant groups ofsmall,unfedleecheswereexposedtoaspotlightfrom behaviors (Tully andQuinn,1985).Inthepresent studies,subject Group trialscanbeeffective waysofexaminingavoidance during rapidshortening. interneuron knowntoreceivevisiblelightinputandbeactive Additionally, ItestedtheinfluenceofUVRonahigher-order receptor neuronsintheleechcouldbeactivatedbyUVR. were directed.Ialsoexaminedwhetherknownvisualpathwaysand and respondtonearUVR,determinedwhethertheresponses of theseopsinshavenotyetbeendetermined. a rhabdomericorigin(Döringetal.,2013).Thespectralsensitivities has recentlybeenshowntoexpressmultipleopsinsconsistentwith Hirudo the segmentalsensilla(Sawyer, 1986).Althoughtheopsinsin not yetbeencharacterized,buthaveassumedhomologousto activity recordedfromnerves.Thesensillaintheheadregionhave which persist(Mann,1962;Sawyer, 1986)andmaycontributetothe head appeartoreplaceseveralofthesegmentalsensilla,some sensory modalities(Jelliesetal.,1994).Thecephaliceyesinthe stereotyped fasciclesintheCNSthatmightcorrespondtodifferent sensilla havealsobeenshowntosegregateintodiscreteandhighly Lasansky andFuortes,1969;Peterson,1984a).Axonsfromthe central nervoussystem(CNS)(FioravantiandFuortes,1972; to lightandproduceactionpotentialsthatareconveyedintothe RESEARCH ARTICLE UVR Leeches showed anegative tonear phototacticresponse RESULTS trial. Asexpected frominspection,theUVR responseswere s other. Neitherrednorbluelightevokedaresponsewithinthe60 to determinewhichmeanswere likelytobedifferent from each analysis (Fisher’s leastsignificantdifference; LSD)wasconducted time, orwiththeinteraction of wavelengthandtime. The two-wayrepeatedmeasures ANOVA confirmed aneffect of In thisstudy, Iassessedtheabilityofmedicinalleechestodetect V ultravioletradiation ultraviolet opticaldensity neutraldensity UVR leastsignificantdifference UV lightemittingdiode OD centralnervoussystem ND artificialpondwater LSD LED CNS APW List of abbreviations List of have yettobedescribed,theglossiphoniidleech ≤ 2B). 0.001) butnosignificanteffect wasassociatedwith Θ ) quantified Post hoc time coursestudied. influences thatmightkeepleechesclumpedtogetheroverthe short the presentstudiesUVRresponsesappearedtoovercome any Torre, 2011). Althoughthisissuecouldbeinvestigatedfurther, in social relationswhengroupsofleechesareconfined(Bisson and interactions betweenpositivethigmotaxis,negativephototaxis and such mixingwouldberandomratherthanselective.Therecan be quadrant asleftitandstayedout.Theseexperimentsassume that example ifasmanyindividualsenteredandremainedinthe would bepossibletomissthebehaviorofindividualleeches,for response in100% oftrialsregardlesswhether theheadortailwas of nineindividualsacrossalltrials, UVRstimulationresultedina 2)individuals.Inbothsets 1)andolderfed (Table unfed (Table generally paralleledthoseofthe subjectgrouptestsforbothyounger to different wavelengths.Theresponsestodifferent wavelengths individual untetheredleecheswere video-recordedduringexposure to examinetheresponseasa functionoffeedingstatusorage, To gaininsightintohowindividual leechesrespondedtolight,and remained significantlydifferent ( from redandblue(P s,bothgreen(†)andwhite(§)lightresponsesweredifferent 45 (*P different fromallotherwavelengthsattimes,except0 has beenpreviouslydescribed(Kretzetal.,1976). (not shown).Theresponsetovisiblelightforbothcephaliceyesandsensilla multiple smallersensillaineachsegmentthatcontainphotoreceptorneurons 1–5 ontheinset).Inadditiontopigmentedcephaliceyesthereare attach totheirsubstrate,andfivepairsofpigmentedcephaliceyes(marked have suckersatanteriorandposteriorendsofthebody, whichtheyuseto Fig. wavelengths of light as predicted by group trials bygroup lightaspredicted wavelengths of Individuals showed anegative phototaxistodifferent

≤ .Medicinalleechesaresegmented worms. 1. 0.001) butwerenotdifferent fromeachotheracrosstime.At The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 ≤ .0)ada 0s,thewhitelightresponse 0.005) andat60 P ≤ 0.005). Inthesegrouptrialsit These worms 975

The Journal of Experimental Biology * way repeatedmeasuresANOVA revealedaneffect ofwavelength( Error bars show±s.e.m.Aindicate anavoidanceorescapefromthetestquadrantsuchthattherewerefewerindividualsattimethan0. two- Shorten+ Shorten tested (legendshowssymbolsassociatedwitheachwavelengthrangetested).Samplevaluesof ecnae1010000 .400 .40.15 4/27 0.04 4/27 1/27 0 0/27 0/27 0/27 0 0/27 0/27 0.04 1/27 1/27 0.04 1/27 0/27 0.04 1/27 1/27 0 0/27 0/27 27/27 100 10/27 27/27 100 0/27 Percentage Total Extend+ Extend 976 RESEARCH ARTICLE a H, head;T, tail. H Response of aquiescentindividual,therewasreleasetheanteriorsucker 3.Whenthespotoflightwasdirectedathead is showninFig. UVR in100%ofthetrialsbothgroups.A setoftypicalresponses leeches. Individualleecheswithdrewthewholebodyawayfrom the both headandtail. expected thatindividualleecheswouldrespondtovisualstimuliat and extendoutofitiftheywerealreadyinit.Itwastherefore seemed toretractfromthequadrantiftheyentereditwiththeirhead, Inspection ofthesubjectgroupvideosrevealedthatindividuals second individual,sothetotalnumberoftrialsforthatsetwas26. individual, andonetrialwasmissedduringtailstimulationina group, asingletrialwasmissedforheadstimulationinone for atotalof54eachfivewavelengthstested).Forthefed making atotalof270responses(27trialsatthehead,27tail (three each,ineachofnineindividuals)forwavelength, larger inthefedgroup.Forunfedgrouptherewere27totaltrials combined). Theresponsefrequenciesforvisiblelightwereslightly applied tothetail(~8%overallforheadandpresentations was appliedtothehead,and11–12% ofthetrialswherelightwas evoked responsesinonlyapproximately4%ofthetrialswherelight stimulated. Incomparison,acrossallindividuals,visiblelight sinthisexample.(B)Theresultsofsubject/groupavoidancetrialsplottedas remained attimes30,45and60 Θ example).Theavoidancefraction, shows thedistributionofleechesattime0.Asterisksindicateindividualscountedasbeingintestquadrant(seventhis Fig. Table depending on whether theheadortailwas stimulateddepending onwhether UVR-evoked avoidance behavior involved responses different Leeches beganto swimorcrawl. P , allowed for comparisons to be made across time and wavelength. At time 15 s there were three individuals in the quadrant, but only asingleindividual stherewerethreeindividualsinthequadrant,but , allowedforcomparisonstobemadeacrosstimeandwavelength.At15 ≤ The responsestoUVRwerecomparableinbothfedandunfed 0.001,

2. LeechesexhibitedstrongUVRavoidanceorescape. 1. Numberandtypeofresponsestophotostimulationhead andtailofunfedindividuals a a § P ≤ 0.005, † 82 /702 /712 /702 /712 0/27 1/27 0/27 0/27 0/27 1/27 0/27 0/27 0/27 18/27 /71/702 /702 /702 /702 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 0/27 17/27 0/27 0/27 9/27 UVR P ≤ 0.005. AB Θ=0.86 Θ=0.57 T Avoidance fraction 1 – Θ=0.86 Θ=0.86 Start no. Time no. H Blue = (A) ForillustrationstillshotswerecapturedfromonetrialofUVRexposure.Thetopleftpanel T P ≤ 0.001), butnottime. Avoidance fraction Θ –0.3 –0.2 –0.1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 0 5 500–600nm 4 615–640nm 3 515–520nm 2 460–470nm 1 395–405nm H Green be resolvedasindividualactionpotentials(Kretzetal.,1976). The overlap witheachothertoproducecomplexwaveformsthatcannot the cephalicnerves.Individuallight-evokedspikesaresmall and Photoreceptor actionpotentialscanbedetectedbyrecording from 2). scored asan‘extend’ response(Table rapid extensionlaterallyawayfromthelight;thisbehaviorwas in arelativelyshortenedstate,headstimulationwithUVRcaused 2).Inadifferent fedindividualthatwas quiescent response (Table contract itsposteriorend;thisbehaviorwasscoredasa‘shorten’ stimulation ofthetailcausedittoreleasesuckerandrapidly individual thatwasquiescentbutinanalreadyextendedstate,UVR 1and2).Inonefed exposure (shorten+andextend+inTables individual oftencrawledorswamformanyminutesfollowingUVR of thebody. Inresponsetobothheadandtailpresentations,the posterior suckerandsubsequentcontractionoftheportion reattachment oftheanteriorsuckerfollowedbyrelease extension oftheheadawayfromstimulus.Thentherewas example), butthisresponsewasalmostalwaysfollowedby sinthis 3P, asterisk;at2.5 release oftheanteriorsucker(Fig. to thetail(Fig. somewhat different responsewasseenwhenstimuliwerepresented 3E,asterisk).A s(Fig. example, theresponsebeganwithin2 followed byashorteningoftheanteriorportionleech.Inthis Cephalic nerves responded tonearUVRstimulation responded Cephalic nerves *

15 Post hoc Post-stimulus time(s) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 T Θ wr acltda 5stimepoints.Positive werecalculatedat15

analysis revealedasignificantdifference betweenmeans, * 3, lowerpanels).Thefirstsignofaresponsewas

30 4560 Θ H Red § againsttimeforeachofthewavelengths * † § T * H White Θ values T

The Journal of Experimental Biology Shorten+ Shorten ecnae101001 .400 .8000 .90.31 8/26 8/26 0.19 5/26 0/26 0.04 1/26 1/26 0 0/26 0/26 0.08 2/26 2/26 0.08 2/26 0/26 0.04 1/26 1/26 0.19 5/26 0/26 26/26 100 5/26 26/26 100 0/26 Percentage Total Extend+ Extend the groupexhibitedawiderangeofresponseproperties,including Over 50individualphotoreceptorsweresampledfromeyes1–4and made inthepresentstudy. to light,butnofurtherassessmentofdifferent cephalicnerveswas smaller DCcephalicnervewastestedandyieldedsimilarresponses 4B–D).Inasinglecase,the as towhite,blueandgreenlight(Fig. 4A,F)aswell DA andDBexhibitedstrongresponses toUVR(Fig. 4E).Theeyesassociatedwith responses toredlightwereseen(Fig. sbetweeneachone.Nonerve and UV, withaperiodof10–15 wavelengths werepresentedintheorder:UV, white,blue,green,red each ofthreestimuliatthesamewavelength.Thedifferent sbetween swith5–6 and notincluded).Lightwaspresentedfor3–5 were sampled(asingleDA nerve was damagedduringdissection 4.Responses fromsixDBandfiveDA nerves is illustratedinFig. very similarresponseswereseen.A representativesetofresponses (DA) and-B(DB)nervesineachofthreeindividualpreparations, how manyphotoreceptorsarerespondingtoanygivenstimulus. the eyestolightbynerverecording,itisnotpossibledetermine the head.Thus,whereasitispossibletodemonstrateresponsesof responses mayalsocontainactivityfromuncharacterizedsensillain a H, head;T, tail. H Response RESEARCH ARTICLE Table stimulation tonear UVR responded Individual photoreceptor Leeches begantoswimorcrawl. In eachoftherecordingsfrombilaterallypaireddorsal-A 2. Numberandtypeofresponsestophotostimulationheadtailfedindividuals a a 42 /652 /622 /602 /652 0/26 5/26 0/26 0/26 0/26 2/26 0/26 5/26 1/26 14/26 /62/602 /602 /602 /602 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 0/26 20/26 0/26 1/26 11/26 UVR T H Blue T H Green photoreceptors examinedsofarrespondedtoUVRover found inthisbriefsurvey. Ratheritwasobservedthatallcephalic different fromthoseforvisiblelight,narrowlytunedcellswerenot appears possiblethatsomereceptorcellsrespondingtoUVRwere UV andslightlyresponsivetoredwavelengths.Thus,althoughit 5C)wasslightlymoreresponsive togreenthaneitherblueor (Fig. green andUV wavelengths,andslightlyresponsivetored.A third 5B)wasaboutequallyresponsivetoblue, red, whereasanother(Fig. UVR thanblueorgreenwavelengths,andnotatallresponsiveto 5A)appearedslightlymoreresponsiveto filters. Oneneuron(Fig. small rangeofluminosityproducedusingneutraldensity(ND) possible tocomparetheresponsesdifferent wavelengthsacrossa By examiningthemaximalpassivedepolarizationproduced,itwas frequency ofactionpotentialsevoked,wasvariableindifferent cells. the magnitudeanddurationofafter-hyperpolarization, andthe parameters suchasthetimeresponselastedbeyondstimulus, 5.Inallcases,lightcausedarapiddepolarization.Response Fig. from threecellsthatrepresentthoseexaminedareillustratedin narrowly tunedtoanyoftheLEDwavelengthranges.Responses appeared thatnoneofthephotoreceptorsexaminedsofarwas LED. Allbutafewcellsexhibitedactionpotentials.Qualitativelyit extents, andanumberalsoshowedweakerresponsestothered examined respondedtoUV, blueandgreenwavelengthstodifferent some thatwerephasicandotherstonic.Allreceptors stimulus (S,T). releasedandtheposteriorendshortenedawayfrom by extensionoftheanteriorbody. Withinafewmoresecondsthetail s(P,*) butthisresponsewasimmediatelyfollowed approximately 2.5 stimulus wasappliedtothetail,anteriorsuckeragainreleasedat anterior suckerreleasedandshorteningwasinitiated.Whenthesame sofheadstimulationwithUVR(E,*),the stimulation (K–T).Within2 sfromaUVRtrialinvolvingheadstimulation(A–J)andtail 0.5 location. or byextendingandthenshortening,dependinguponstimulus Fig.

.Individualsremovedthemselvesfrom UVRbyshortening, 3. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 T Screen shotsfromthedigitalvideosweregrabbedevery H Red T H White T 977

The Journal of Experimental Biology 978 RESEARCH ARTICLE than thatevokedbyblueorred light( response atfullstrength(0OD) andtheseresponsesweregreater the greenandUV LEDs.Theyeachevokedacomparable S-cell 7A)werefrom The mosteffective stimuliappliedtothehead(Fig. conducted todeterminewhichmeans weredifferent fromeach other. density (OD;P revealed asignificanteffect forbothcoloroflightandoptical 7).TherepeatedmeasuresANOVA range ofluminosity(Fig. quantitatively acrossred,green,blueandUV light,alsoacrossa stimulus, responsesatbothheadandtailwerecompared were consistentwithexpectationsforaphysiologicallyrelevant and greenlight.To determine whethertheS-cellresponsestoUVR msrange,similartothoseobservedforresponses blue 100–200 from UVRstimulusonsettofirstactionpotentialwerein the presented totheheadortail,inallleechesexamined.Alllatencies shown inFig. response toredlight,whentheyoccurred,wasalsolonger(example S-cell responsetoredlight(Fig. pathway (Kretzetal.,1976).Therewasmorelimitedandvariable was consistentwithphoto-activationoftheS-cellfast-conducting cmfromthehead andtail.Thisveryshortdelay electrode was2–3 ms.Therecording potential wasshort,approximately100–200 (Fig. and bluelightevokedburstsofactionpotentialsthatadaptedrapidly responses. Asexpected(Kretzetal.,1976;Sahley1994)green shown). However, whentheS-cellwasexamined,itshowedstrong obtained fromtheseneuronstoanywavelengthrangeusedhere(not excitor (HE)motorneuron(Mulleretal.,1981).Noresponseswere mechanosensory neurons(T, P andN)wasimpaled,astheheart 6).Intwoexperimental preparationseachofthemajor or tail(Fig. used totestneuronalresponseslightstimuliappliedatthehead with neuronalsomataimpaledinmidbodyganglion9,10or11 was those earlierstudies(Kretzetal.,1976).A semi-intactpreparation response propertiestothecephaliceyes,butUVRwasnottestedin demonstrated thatthesegmentalreceptorshaveverysimilar response suggestedthatsensillaalsorespondedtoUVR.Kretzetal. The observationthatlightstimulionthetailcanevokeanescape to visiblelight. physiologically relevantrangesofluminosity, similartotheresponse most effective stimulusattheheadwas green LED( A higher-order interneuron responded toUVR responded interneuron A higher-order EF DE A The S-cellrespondedvigorouslytostimulationbyUVR(Fig. 6), andthelatencyfromstimulusonsettofirstaction Green UVR 6). ≤ 0.001). Post hoc BC

6). ThelatencyofS-cellspikesin analysis (Fisher’s LSD)was White Red † P ≤ .0) t03OD,the 0.005). At0.3 † P ≤ 0.005).

6) Blue UVR in responsefromoneODfiltertothenext( expected, inmanycasesforagivencolor, therewasalsoareduction UVR ( ODwas 7B),themosteffective stimulusat0 For tailstimuli(Fig. behavioral responses werefoundinresponse tovisiblelight, examine theiruntetheredresponses. Asexpected,fewormuted Leeches wereexposedtolight bothindividuallyandingroups,to conditions mayuseitasanappetitive cue. leeches arenotparticularlyaverse tovisiblelight,andundersome Harley etal.,2013).Thus,itmightbesuggestedthathirudiniid disturbances) toorientandlocatepotentialprey(Harleyetal., 2011; with tactilevibration(bothofwhicharerelatedto water frequency-modulated (movingbars)visiblelightincombination recent setofelegantexperimentshaveshownthatleeches use (Dickinson andLent,1984;CarltonMcVean, 1993),anda leeches orienttomovingbarsofvisiblelightwhenhungry shadows, orbrief,brightflashes(Kretzetal.,1976).Inaddition, visible spectrumtoorientwhileswimming,andtheyrespond to neuronal response(J.J.,unpublisheddata).Leechesalsouse the nmevokednobehavioralor emission peaksatapproximately850 heat (DickinsonandLent,1984).IconfirmedthatIRLEDs with (IR) (Harleyetal.,2011) althoughtheycandetectandrespondto (Kretz etal.,1976).Leechesappeartobeinsensitivenearinfrared nm to violetandredlight.Theshortestwavelengthtestedwas430 showed thatthesensorycellshavealower(butnotzero)response nm(Kretzetal.,1976).Theyalso responses between500and600 nm(green)with substantialneuronal sensillar photoreceptorsis540 that themidrangepeakofspectralsensitivitybothcephalicand light weresimilartothoseinpreviousreports(Kretzetal.,1976). cell) areallactivatedbyUVR.Theresponsesobservedtovisible individual photoreceptorneuronsandahigher-order (theS- stimulus wasapplied.Inaddition,Ifoundthatcephaliceyes, removed aleechfromUVRregardlessofwhereonthebody coordinated negativephototacticresponsetoUVR;thisbehavior These resultsshowthatmedicinalleecheshavearobustand other wavelengths,atallODsthan0(* effective atbothheadandtail,beingsignificantlyweakerthanall Leeches to UVR responded DISCUSSION most effective stimulus( In anextensivestudyofleechvisualcells,Kretzetal.determined 5 s † P ≤ .0)weeswt h . ODfilterthegreenwas 0.005) whereaswiththe0.3 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 (F) SecondexposureresponsetoUVR. light. (D)Responsetogreen(E)red UVR. (B)Responsetowhitelight.(C)blue the lighton(downward)andoff (upward).(A)Responseto was alsoaninstantaneoustransientassociatedwithturning were deliveredatsaturatingintensities.Ineachrecordthere each trace)indicatesthetimingoflightstimulusandall 1976). Theoutputfromaphototransistor(shownbelow were complexandslowlyadaptingasexpected(Kretzetal., hand forafewseconds.Massedphotoreceptorresponses from theLEDwandswasdirectedatheadandheldby been severedfromthesupra-esophagealganglion.Light placed uponthecutendofDBcephalicnervethathad a rangeofvisiblewavelengths. Fig.

.GroupsofphotoreceptorsrespondedtoUVRand 4. † P ≤ 0.005). Redstimuliweretheleast § A suctionelectrodewas P P ≤ ≤ 0.001). Asmightbe 0.005).

The Journal of Experimental Biology Green Green swimming and crawling (BrodfuehrerandFriesen, 1984;Lentand these transmittersystemsstrongly interactwithfeedingbehavior, Kristan andNusbaum,1982-1983; PuhlandMesce,2008) are regulatedbyamines(Crisp andMesce,2006;Eschetal.,2002; influencing motivation,behavioral choiceandresponsestostimuli and Kristan,2010;Miselletal., 1998).Manyoftheinteractions evoking certainbehaviors,such asswimmingorcrawling(Gaudry responses (GaudryandKristan,2009)theprobability of been wellestablishedthatsatietystronglyinfluencessensory show adifferent responseto UVRthantheunfedleeches.Ithas 1,2). 3,Tables recently fedindividuals(Fig. 2)andindividualanimals as wellinunfedand grouped (Fig. These UVRresponsesoccurredin100%ofthetrials, both responses wereevokedwhentheleechesexposedtoUVR. especially redorblueLEDs.Incontrast,robustavoidanceescape RESEARCH ARTICLE Green C B A UVR UVR UVR Blue Blue Blue Red Red Red I includedthegroupoffedindividualsexpectingthattheymight 100% T 100% T 100% T 25% T 5% T 5% T 10 mV 10 mV 10 mV 0.25% T 5% T 1% T 2 s 2 s 2 s

Passive depolarization (mV) 10 20 30 10 20 30 10 20 30 |log % Transmittance| 1.0 2.03.0 1.0 2.03.0 1.0 2.03.0 light. Thus,stimuli detectedbythecephaliceyes andpossiblythe responses) andthenreleasedthe tailsuckertowithdrawfromthe first extendedthehead(which involvesanoppositesetofmotor occurred, whereaswhenthetail wasexposedtoUVR,theleeches was exposedtoUVR,withdrawal oftheheadbybodyshortening The responsestoUVRwerecomplex anddirected.Whenthehead leeches. to UVRofunfedleecheswasthesame(100%)asthose fed negative phototaxistoUVR.Instead,thefrequencyofresponses 2), Iexpectedthatunfedindividualsmightnotexhibitasstrong a 1, the resultsforvisiblelightinthesestudiesaswell;seeTables expected tobelessaversevisiblelightthanfed(consistent with Dickinson, 1984;Willard, 1981).Since unfed individualswere integrated to produce avoidance toproduce orescape integrated UVR-evoked behavior involved distinctmotor actions The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 to eitherblueorUVwavelengths. response togreenremainedmorerobustthan in B.Yet, withdecreasedluminositythe to redlightsimilarthatinthereceptorshown the neuronshownheretherewasaresponse decreased withluminosity. (C)In green andUVwavelengths,thisresponse cell seemedtohaveanequalresponseblue, ODthe fell off withdecreasedluminosity. At0 100% transmittance|),buttheresponsequickly OD(|log there wasaresponsetoredat0 than theothers.(B)Inneuronshownhere the responsetoUVRremainedmorerobust highest luminosity. Asluminositydecreased, responses toblue,greenandUVlightatthe at anyluminosity, andapparentlyequivalent shown heretherewasnoresponsetoredlight strong responsestoUVR.(A)Intheneuron (including thethreeshownhere),therewere Arrowheads indicatestimulusonset.Inallcells mV. these threeexampleswere–35to–40 intracellular electrode.Therestingpotentialsin different leechesrecordedusingasharp eye 2(A),3(B)and(C)fromthree different primaryphotoreceptorneuronsfrom visible light. neurons respondedtoultravioletand Fig.

.Individualprimaryphotoreceptor 5. Activity fromeachofthree 979

The Journal of Experimental Biology 980 RESEARCH ARTICLE work, buttheresponseofleechestolightshoneupontail(Fig. tail (Fig. stimuli transducedbythesegmentalsensillaofbodywalland anterior sensillamustactivatesomewhatdifferent CNScircuitsthan expression ofthe behavior(ShawandKristan, 1999;Arisietal., (Sahley etal.,1994),itisneither necessarynorsufficient for Kristan, 1995),andisrequired forsensitizationoftheresponse conducting pathwayisactivated duringshortening(Shawand 1982; ShawandKristan,1995). AlthoughtheS-celloffast- to theanteriorportionofleech, itshortensrapidly(Kristanetal., Pelligrino, 1978).Whentactile stimuli orelectricshocksareapplied when stimulationisappliedtotheanteriorbody(Magni and 1988). A similarbehaviorhasbeenwelldocumentedinleeches (Drewes andFourtner, 1989;ZoranandDrewes, 1987;Zoranetal., 2010). terminate behaviors,swimorcrawl(MesceandPierce-Shimomura, proprioceptive inputssimilartothatdescribedforthedecision to This resultimpliesahighdegreeofintegrationexternal and remove itfromtheUVR,whateverstartingconditionwas in. to UVR.Theleechrespondedinwhateverfashionwasneeded to 6,7)suggeststheyaresensitive as welltheS-cellresponses(Figs Cephalic eyes Rapid withdrawalhasbeenextensivelystudiedinoligochaetes

3). Individualsensillawerenotdirectlyexaminedinthis tmlt edStimulatetail Stimulate head xrclua lcrd Intracellularelectrode Extracellular electrode 50 mV Green EC EC EC EC IC IC IC IC Blue UVR Red S10 S10 S10 S10 7-8 7-8 7-8 7-8 500 ms Sensilla

3) multiple, widely spacedsensillarinputs,orboth. in different locations,ordifferent degreesofconvergence of neural responsesmightbetheresult ofdifferent receptorproperties head. Itremainstobedetermined whetherthesebehavioraland 12% ofthetimecomparedwith only4%whenpresentedatthe 2) wherevisiblelighttothetail evokedaresponseapproximately 1, with thebehavioralresponsesseen inindividualleeches(Tables evoked amorerobustresponsegenerally, thiswouldbeconsistent 7).Iftailstimulation to tailstimulationseemedshiftedupward(Fig. wavelengths wereexaminedacrossluminosity, theoverallresponse asymmetric. Forexample,whentheresponsestoUV, blueandgreen 7B)arepossiblyinvolvedbecausetheyappeartobe tail (Fig. 7A)and characterized. TheS-cellresponsestolightatthehead(Fig. responses dependinguponstimuluslocationremainsto be escape. 6,7),butnotrevealingthedetailsofhowUVRevokes UVR (Figs interneurons involvedinrapidmovementscanbeinfluenced by strongly activatedbyUVRexposure,confirmingthathigher-order 1999; Arisietal.,2001).Inthecurrentstudies,S-cellwas appears tounderliethewhole-bodyshortening(ShawandKristan, 2001). Rather, thereisaparallelnetworkofinterneuronsthat The light-activatedcircuitrythatunderliesdifferential behavioral The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 intracellular (IC)actionpotential. the largestmultiphasicspikethatcouldberelated1:1with always distinguishableintheextracellular(EC)recordingsas longer latencyfromstimulusonset.TheseS-cellspikeswere instances wheretheredLEDevokedresponses,thesewere stimulation usingUV, blueandgreenLEDs.Inthose cell producedshortlatencyactionpotentialsinresponseto active, butgenerallysilentuntilstimulatedwithlight.TheS- mVandthecellwassporadically was approximately–50 exposed forimpalement,ifnecessary. Therestingpotential in midbodyganglion10.Ganglia9and11 wereroutinely Arrowheads indicatestimulusonset.TheS-cellwasimpaled extracellular (EC description. RepresentativeS-cellintracellular(IC head totail.SeeMaterialsandmethodsforadetailed It conservedcontinuityoftheCNSandallmajornervesfrom intact preparationwasusedtoevaluateneuronalresponses. light stimulationatboththeheadandtail. pathway, respondedvigorously and withshortlatencyto Fig.

.TheS-cell,aninterneuronofthefastconducting 6. 7-8 ) voltagerecordingsareshown. A semi- S10 ) and

The Journal of Experimental Biology transduce theUVR andgeneratediscreteinputs forUV andvisible Yamaguchi etal.,2010).Itremainstobedetermined howleeches a subsetofphotoreceptorsinthe ommatidia(EarlandBritt,2006; 2001), andDrosophila including avoidanceofambient UVR(LeechandWilliamson, UVR (SmithandMacagno,1990) andexhibitsadaptiveresponses, Daphnia (Jacobs, 1992)andinvertebrates(Salcedoetal.,2003).Forexample, discrete UVRreceptorcellsarealsowellknowninbothvertebrates to generateUV-tuned neuralinputs.UV-selective photopigmentsand screening pigments(Honkavaaraetal.,2002;Jacobs,1992)in order behavioral responsestothem.Manyanimalsuseoildroplets or between UV andvisiblelight becauseitcangeneratedifferential wavelengths. 7B)followedbyhighsensitivitytogreen decreased luminosity(Fig. sensitivity toUVRatthehighestintensitythatfelloff with stimulation wereconsistentwiththisaswell,maximal spectrum andanadditionalpeakforUVR.Theresponses tail have twopeaks,theexpectedoneingreenregionof 7A).Thiswouldbeconsistentwithresponsesthat strength (Fig. stimuli wereequallysensitivetoUVRandgreenlightdeliveredfull convey thatinformationtotheCNS.TheS-cellresponseshead leech visualsystemhastheabilitytoeffectively transduceUVRand 5)clearlyshowedthatthe The responsesofindividualneurons(Fig. primary photoreceptorneuronswhilestimulatingthemwithlight. was beingdetected,Ialsorecordedfromaselectionofindividual 4E).To directlyassesshowUVR illumination bytheredLED(Fig. 4A–D,F),whereasnoresponsewasdetectedduring LEDs (Fig. that wascomparabletoproducedbyblue,greenandwhitelight 1 and2exhibitedavigoroussustainedresponsetoUVR,one Cephalic nerverecordingsrevealedthatthephotoreceptorsofeyes † Fig. RESEARCH ARTICLE revealed aneffect ofwavelength luminosities comparedwiththesamestimulipresentedatheadoverrangeof|log%transmittance|.A ANOVA two-wayrepeated-measures light attenuatedasitdidforheadstimulation.TheresponsestoUV, higheracrossdecreased blueandgreenwavelengthstimulationonthetailallremained green light.Theresponsetored with theheadstimuliUVRresponseattenuatedinitialdecreaseinluminositymoresothandidto consistentresponse,butas the tailwerealsoabletoevokestrongS-cellresponses.At0|log%transmittance|UVRclearlyevokedmostrobustand (0.3 OD),whereasallothersdecreasedsignificantly. Theresponsetoredlightattenuatedmostreadilywithdecreasedluminosity. (B)Lightstimulipresentedto plotted asOD=|log%transmittance|,theresponsefelloff rapidlybutdifferentially. Green lightremainedaneffective withslightlydecreasedluminosity stimulus luminosity,head evokedstrongresponsesfromtheS-cell,withmostrobustresponseequallybygreenandUVwavelengths.Withdecreased UVR-activated photoreceptors P ≤ In someway, yettobedetermined,theleechisabledistinguish 0.005 aswellwithinwavelengthbetweenOD

.TheS-cellrespondedstrongly, butdifferentiallytovisibleandUVRstimulipresentedboththeheadtail. 7. has beenshowntohaveindividual ommatidiasensitiveto has awell-describedUVRsense mediatedby Normalized S-cell post-stimulus frequency 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 P A ≤ 0.001, andOD 0 † † * § †,§ §

0.5 ,§ Stimulate head * § P § ,§ ≤ P 0.001. Posthocanalysisrevealedasignificantdifference betweenmeansatagivenOD,* ≤

0.005. 1.0 1.5 § * Red Green Blue UVR |log % Transmittance|

2.0 * 60×15 werereleasedfromtheir cocoonsat USA), weremaintainedinabreeding colonyatroomtemperature(20°C). Leeches USA (Westbury, NY, USA)andNiagaraLeeches(Cheyenne,WY, Medicinal leeches, adaptive behavior. by thephotoreceptorsandintegratednervoussystem for laboratory, butitremainstobedeterminedhowUVRistransduced of theanimals.LeechesrespondvigorouslytoUVRin the leeches andmayalsoplayanimportantroleinthenaturalbehavior be apreviouslyunder-appreciated factorinthe distribution of Differential UVRpenetrance innaturalaquaticenvironmentsmay potentially threateningconditionsinthenaturalenvironment. avoid environmentswithhighUVRexposure,ortoescape from phototactic responsetoUVRwouldeffectively permitleechesto responses tovisiblelight.Thereforeitisprobablethatthenegative to UVR.Thisresponsewasevenmorereliablyevokedthan a discrete,robustandcoordinatedavoidanceand/orescaperesponse distinguish thesepossibilities. peak responsebiasedtowardUVR?Thepresentresultscannot the UVRresponseencodedbypopulationsofreceptorswith yet, undiscoveredreceptorcellsthataretightlytunedtoUVRoris exhibit absorptionpeaksintheUV andgreenregions?Arethere,as opsins inHirudo.Thisremainstobedetermined.Doallreceptors expression (Döringetal.,2013).Perhapstherearealsomultiple leech photopigment withmultipleabsorptionpeaks?Recentworkthe expressed indifferent amountsindifferent cellsoristhere asingle light. Forexample,aretheredifferent photopigmentsthatmightbe Preparations MATERIALS ANDMETHODS These studiesshowforthefirsttimethatmedicinalleecheshave 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 B mm Petridisheswith artificialpondwater[APW: 0.05%(w/v)Instant Helobdella 0 † The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 * † §

,§ 0.5 Hirudo * Stimulate tail has revealedmultipleformsofopsingene ,§

spp. (Siddalletal.,2007),originally obtainedfrom 1.0 1.5 * ,§ (A) Lightstimulipresentedtothe

2.0 * ≥ 30 daysandwerekeptin P ≤ 0.001, 981

The Journal of Experimental Biology scale. globally adjustedtobalancebrightnessandcontrastconvertedgray Photoshop CS3(AdobeSystemsInc.,SanJose,CA,USA).Imageswere sintervalsandcroppedin sor5 individual screenshotsweregrabbedat0.5 MacBook Prolaptop(Apple,Cupertino,CA,USA).To generatethefigures, test surface.Videos werereviewedframebyusingQuickTime 7ona 982 RESEARCH ARTICLE (395–405 20–25 was aT-1 3/4packagewithaclear, roundlensandaviewingangleof Hand-held wandsdirectedthelightfromLEDstotestsurface.EachLED of 400.Thevideocamerawasmountedonasmalltripod30 used torecordalltrialsusingthehighestsensitivityandaneffective ASA being 1.5 prepared byprintingahexagonalgraphpaperarrayonwhitepaper, eachunit the benchsurface(LuxMeterProiPad2application).A testsurfacewas lxat area. Thisresultedinadimbackgroundilluminationmeasuredas5–10 two 15 with diffuse backgroundlightingmaintainedbydirectingadesklampwith Untethered leecheswereexposedtodifferent conditionsinanisolatedroom individual 60×15 breeding colony. Bothsubject/groupsandindividualswerehousedin and wereselectedatrandomfromagroupofapproximately200inthe were keptinseparatedishesbetweentrials.Theninefedleechesolder three individualswereselectedfromeachcohort.The a different setofthreecocoonsthathadbeenkeptinindividualdishes,and numbers ofsiblings. as fivesubjectsfortesting.Notethatindividualcocoonshavevariable cocoons wereusedtoestablishthesefivegroups,whichthentreated four different siblinggroups(fourdifferent cocoons/group).A totalof10 individuals thatwereselectedbycombiningfivefromeachof and 50 months oldwereusedinthisstudy. Theunfedjuvenilesweighedbetween30 daysofage(juvenile)andtwice-fedanimals(nineindividuals)6-to9- 75–90 water]. Bothunfedanimals(109individualsfrom13siblingcohorts)at Ocean seasalt(SpectrumBrandsInc.,Madison,WI,USA)dilutedinspring LED stimuli Behavioral arena

Phototransistor response The ninejuvenileunfedleechesobservedindividuallywereselectedfrom juvenilesweredividedintofivegroupsof20unfed For grouptrials,100 (mV) B A 10 20 30 40 50 deg. Thefivedifferent nominalwavelengthsoftheLEDswere:UV g e ece ege ewe .5ad08g. mg, fedleechesweighedbetween0.45and0.8 W fluorescenttubestowardthewallpointingawayfromtesting 0 nm), blue(460–470 mm indiameter. A Vivitar digitalvideocamera(508NHD)was Direct Reflection 2 10

mm FalconplasticPetridishesatroomtemperature.

12 nm), green(515–520 Distance fromdetector (cm)

14 1618 V leGenRdWhite Red Green Blue UVR nm), red(615–640

20 cm abovethe

22 nm) White Red Green Blue UVR

24 Each groupreceivedthreerepeatedtrialsofallfivewavelengths.Time 0 random sequencesthatdeterminedtheorderofpresentationwavelengths. stimulus wasthenpresentedusingadifferent wavelength. min.Thenext minandneverlongerthan30 which typicallytook2–5 pipette todetachthemfromthesubstrate;theywerethenallowedsettle, was taken.Betweentrials,theanimalsweregentlystirredwithaplastic s,asavideorecording example). Thelightwasheldinplaceforatleast60 and minimizevisibleilluminationofadjacentquadrants(seeFig. that necessarytocenterthebeamofilluminationonquadrant(3–4 leeches inaquadrantwasfourto12.Thedistancepredeterminedbe 0), andthentheLEDwasturnedon.Therangeofstartingnumbers selected withatleastfourindividualsinit(toavoidstartingconditions were quiescent.AnLEDwasheldaboveaquadrantthatarbitrarily min,whenmostanimals leeches wereallowedtosettleforapproximately10 were leftonforthesestudiestokeeptheanimalsinsidedishes.The group wasplacedontopofthequadrant-markedsurface.Theplasticlids For grouptrials,aquadrantwasdrawnonthetestsurface.Eachsubject 380–385 preparation (notshown)includingonewithemissionat395–405 other sourcesweretestedonunfedjuvenilesandthesemi-intact (through eBaystore,Shenzen,Chongqing,China).UVRLEDsfromthree and thebluegreenLEDswereobtainedfromLongxiElectronics were obtainedfromDave’s LEDHut(througheBayStore,NewMexico) and white(500–600 a 300 V through reported here.GivensmallvariationsinforwardLEDvoltage,9 neuronal responsessimilartotheUVRLEDusedacquiredata Group trials Group the emissionsfromallLEDswereroughlyequal(Fig. phototransistor responsesensitivity. Thesemeasuresofresponseshowedthat including theLEDviewinganglesandintensityaswell measures ofphototransistoroutputwereafunctionseveralfactors current throughafixedresistance(JelliesandKueh,2012).Therelative diameter fiberoptictogatherlight.Thephototransistorwasarrangedgate mm each emissionwasassessedusingaphototransistorcoupledto2 their projectedspotsandreflectedimageswerephotographed(Fig. effort tomeasuretheapproximatestrengthsofstimulifromallfiveLEDs, 8A)thatsaturatedthecamera.Inan LEDs allproducedspotsoflight(Fig. mA.The mA andwhite19 mA,red21 mA,green19 mA,blue19 UVR 19 An onlinerandomsequencegenerator(random.org) producedfive Ω resistoryieldedthefollowingnominalcurrentdrivingeachLED: light begantofalloff. cmtheresponsestoUV, blueandwhite began tofalloff, thenat18 cm,theresponsestoredandgreenwavelengths cm.At14 out to12 by afixedamount.Allwavelengthsproducedsaturatingresponses Kueh, 2012).Eachspotwasthenmovedawayfromthetransducer coupled totheoutputofageneralIRphototransistor(Jelliesand mmdiameterfiber optic faceofatransducer pointed directlyatthe2 exceptionally strongorweakcomparedtotheothers,eachwas examine relativestimulusstrengthandconfirmthatnoLEDwas images ofeachspot,butatagreatlyreducedluminance.(B)To behind theprojectedspotstosimultaneouslyviewreflected in thedigitalimagesoaperpendicularplasticsurfacewasplaced surface tophotographtheemissionspots.Thespotsweresaturated cmabovethetest hole drilledthroughitandpointeddownward,2–3 illumination areas. Fig. nm andoneat360–365

The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 .LEDstimuliwerecomparablystrong overtheprojected 8.

nm). TheUV (referredtoasUVR),redandwhiteLEDs (A) Eachwandwasheldinaplasticbarwith

nm; allofthemevokedbehavioraland

8B).

nm, oneat

8A) and 2 foran

cm)

The Journal of Experimental Biology uniquely determinedbyanonlinerandomsequencegenerator(random.org). order ofpresentationwavelengthsforeachthe18individualswas delivered first,followedbythesamesetofstimulitotail.The s.Stimulitothehead(threetrialsforeachwavelength)were least 30 min.To receiveatrialtheleechhadtoremainquiescentforat as long30 min,butrarelywas allowed tobecomequiescent,whichtypicallytook2–5 115 NaCl,4KCl,1.8CaCl darkness) asdescribedaboveforbehavior. electrophysiology experimentswerecarriedoutindimroomlight(nottotal 5.4 Tris base,andhadapHof7.4(NichollsBaylor, 1968).All this resistance.For subsequentneuronalrecordingscomparing theresponses was placed.A secondchannelofthePowerLab monitoredthevoltageacross (Jellies andKueh,2012)wasplaced justbelowwheretheanteriorsucker k optic-coupled phototransistorthat gatedcurrentthrougha10 mmdiameterfiber s.To monitorpresentationofthelight,a2 sucker for3–6 all ofwhichyieldedsimilarresponses. DA andDBrecordingswereobtainedfrom eachofthethreepreparations, kHz.Multiple (ADInstruments, ColoradoSprings,CO,USA)samplingat10 Tektronix oscilloscopeandcapturedbyanADIPowerLab 26/T Hz.Thetraces weresimultaneouslyviewedona high endsetat1000 Hzand amplifier withthegainsetat1000or10,000×,lowendfilter10 signals wereamplifiedusinganA-MSystemsmodel1700differential with otheraxons(Kretzetal.,1976). carries axonsarisingfromeye1andtheDB2, along and alengthwascleanedliftedfromthetissuesurroundingit.The DA surgically exposed.EachDA and DBwascutnearwhereitjoinedtheCNS 1976) radiatingfromtheundersideofsupraesophagealganglion were main trunksofthetwolarge cephalicnerves(DA andDB)(Kretzetal., mmlayerofclearSylgardresininit.The mmplasticdishwitha3 into a60 four midbodysegmentswasseparatedfromtherestofleechandpinned To recordeyeresponsesfromcephalicnerves,theheadattachedtofirst with ice-coldRinger’s solution.TheleechRinger’s contained (inmmol To prepareananimalfordissectionandrecording,itwasfirstanesthetized crawling (shorten+,extend+). s,notablysubsequentswimmingor other behaviorbeganwithin2–3 either shortening(shorten)orextending(extend).Notewasmadeifany s,movementof thebodywasclassifiedas response wasinitiatedwithin5 s,itwasscoredas0(noresponse).Ifa response wasinitiatedwithin5 sifaresponsewasinitiated.Ifno The stimuluswasremovedbefore5 subsequent analysis. individuals inside.Videos weremadeofheadortailpresentationtrialsfor proved unnecessarytohavethelidsittingdirectlyondishkeepthese for stimuluspresentationinordertofollowsubjectsatanyposition.It untethered testssoconsiderablefreedomofmovementhadtobemaintained surface tostandardizehowclosetheLEDwasplaced.Thesewereall contained a5 individuals weretransferredintoa100 responses toilluminationonboththeheadandtail.Fortesting, Each ofnineunfedandfedindividualswasalsotestedfortheir 0 (Fig. number ofanimalsinthequadrantattimexdividedbythosepresent were reportedasanavoidancefraction( average ofeachthethreeresponsestowavelengths.Results than onequadrant.Theresponseofeachgroupwasthendeterminedasthe that definedthequadrant,sosameanimalcouldbecountedinmore defined asbeinginthequadrantifanyportionofitsbodycoveredline s.Anindividualwas test quadrantwasdeterminedat15,30,45and60 was definedasjustbeforeilluminationandthenumberofindividualsin RESEARCH ARTICLE Cephalic nerverecording Electrophysiology Individual animaltrials The plasticlidofthe100 Each LEDwaspresentedinarbitrary ordertoilluminatetheanterior A suctionelectrodewasplacedonthecutendofacephalicnerveand s. A trial consistedoflightbeingshoneuponeithertheheadortailfor2–5 2A).

mm depthofAPW (approximately30 2 , 1.5MgCl

mm Petridishwasheld3.5 2 , 10D-glucose,4.6Tris maleateand

mm plasticFalconPetridishthat Θ ), whichwasoneminusthe

ml). Eachleechwas

cm abovethetest Ω resistor

l − 1 ) present study. Instead,the S-cell wasusedtoexaminetheeffects ofUVRin a surveyofUVR effects onneuronsgenerally wasbeyondthescopeof 1994). Althoughseveralasyetunidentified cellsshowedresponsestolight, potentials thatadaptedrapidly(Gardner-Medwin etal.,1973;Sahley ildwt mol filled with1 M 1–4 wereobtainedusingglassmicroelectrodes(resistancesof25–45 were clearlyvisibleassphericalcells. When viewedwithobliquelightingtheindividualphotoreceptorsomata pigmented eyecupwascarefullyremovedusingmicro-scissorsandforceps. up intherecordingdish.Thetransparentepithelialcoveringovereach the anteriorsuckerwasdissectedawayfromleechandpinneddorsalside Lasansky andFuortes,1969;Peterson,1984a).Briefly, thedorsalmargin of as describedelsewhere(FioravantiandFuortes,1972;Kretzetal.,1976; To recordfromindividualcephalicphotoreceptors,theeyeswereprepared green LEDs. very broademissionspectrumthatoverlappeddeliveredbytheblueand to different LEDemissions,thewhitewasomittedbecauseithada a small(<20 1981) aswellunidentifiedneurons. TheS-cellwasreadilyidentifiableas stimuli, includingthemechanoreceptors andHEneurons(Mulleretal., were obtainedfromeachoftwopreparations duringpresentationoflight diminishing intensity. Multiplerecordingsfromvariousidentifiedneurons anterior orposteriorsuckerandtheadjacentthreetofoursegments with were obtainedasdescribedabove.LightbeamsfromLEDscovered the lost. be usedtoquantifyS-cellresponseseveniftheintracellularimpalement was was identifiedby1:1correlationwiththeintracellularrecord,itcould then similar tothemethodsdescribedabove.Oncelargest extracellularspike electrode opening.Recordingswereobtained central portioncontainingFaivre’s nerve(Fernandez,1978)wasbeneaththe on theintersegmentalconnectivebetweensegments7and8suchthat the described byKretzetal.(Kretzal.,1976).A suctionelectrodewasplaced were madetoexposegangliainsegments7–11 inmuchthesamewayas midbody segmentswerepinnedventralupward(Fig. were twistedandpinnedwiththedorsalsideorientedupward leeches usedwere5–6 of stimuli,theleechwasdissectedusingcoldRingerasdescribedabove.All To recordfromindividualmidbodyganglionicneuronsduringpresentation selected forpresentation. representative cells(fromeye2fromtwodifferent leechesandeye3)were ND filtersinadditiontousingnofilter. Asaresult,recordingsfromthree to presentred,green,blueandUVRLEDs,acrossatleasttwointerposed able toobtainstablerecordingsineightcellsfortheextendedtimerequired characterization willbethetopicoffuturestudies.ForthesestudiesIwas leech receptors(seePeterson,1984a)]andamorecompletequantitative cells todifferent LEDs[consistentwithpreviousstudiesonvisiblelightand However, there was considerablevariabilityintheresponsesofdifferent studies, over50cellsweresuccessfullyimpaledandstimulatedwithlight. two filterswerestackedtoyieldintermediateorhigherOD.Forthese preparation. Inmostcases,singlefilterswereinterposed,butinafewcases allowing theNDfilterstobeinterposedbetweenlightand insulation thatcouldholdanLEDwand,andwhichhadaslitcutintoit Loveland, CO,USA).A simpleholderwasfashionedfromstiff foampipe wavelengths. TheywereobtainedfromanonlineeBayvendor(caprice47, chromium-coated fusedsilica(7980)suitableforbothUV andvisible density (ND)filterswasused.Thewere25.4 times. To examineresponsesacrossluminositylevelsasetoffourneutral mV andwereheldinstablepenetrationsfor variable range of–35to–48 kHz.Allcellshadrestingmembranepotentialsinthe CO, USA)at10or20 PowerLab 26/T oraPowerLab4/35(ADInstruments,ColoradoSprings, were simultaneouslyviewedonaTektronix oscilloscopeanddigitizedbya (World PrecisionInstruments,Sarasota, FL,USA;773preamplifier).Signals Intracellular photoreceptorrecording Simultaneous intracellularandextracellularrecording Intracellular recordingsfromindividualprimaryreceptorneuronsineyes Intracellular recordingsfromindividualneuronsinganglion9,10or 11 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 μ m) somainthecentralpacket,with fast,overshootingaction

l − 1 potassium acetate)usingastandardrecordingamplifier

cm longwhendissected.Theheadandtailsuckers en passant

mm indiameter;nickel-

6). Smallincisions but otherwisewere 983 Ω ,

The Journal of Experimental Biology oig .J,Leh .M,Wlimo,C . ok,S n ors L. Torres, and S. Cooke, C.E., Williamson, D.M., Leech, W. J., Boeing, A.R. Webb, and M. Blumthaler, rduhe,P .adFisn W. O. Friesen, and P. D. Brodfuehrer, V. Torre, and G. Bisson, 984 RESEARCH ARTICLE in PMCforreleaseafter6months. Undergraduates Grant,no.DBI-1062883,NationalScienceFoundation.Deposited Program [grantnumber52006962];andaResearchExperiencesfor Howard HughesMedicalInstitutethroughtheUndergraduateScienceEducation [grant numberW2013-007];agranttoWestern MichiganUniversityfromthe from theOffice oftheVice PresidentforResearch,Western MichiganUniversity This workwassupportedbyaFacultyResearchandCreativeActivitiesAward The authordeclaresnocompetingfinancialinterests. many helpfulcommentsandeditorialsuggestions. manuscript, andWilliamB.Kristan,Jrforreadingthemanuscriptoffering also thankDanielKuehforhisadviceonstatisticsandcommentsadraftofthe in gatheringinitialelectrophysiologydataandtheirleechhusbandryassistance.I I thankRachelKing,ChristopherRouschandGerardoZayasfortheirassistance All statisticalanalysesontheavoidancefraction( for thatwavelengthrangeandOD. the stimulusonset,normalizedtopeakfrequencywithinthatindividual msafter Responses werecomputedasthespikingfrequencyinfirst500 before thelongerintervalinstimuluspresentationstoallowrecovery. recordings (J.J.,unpublished).ItseemedprudenttoplacetheUVRstimulus responses incephalicphotoreceptors,fromtheS-cellsandsensillarnerve revealed thattheUVRstimulussometimesevokedlong-lastingtonic filters interposed.Thisorderwasselectedbecausepreliminarystudies were presentedintheorderred,green,blueandUV, withandwithoutND OD). Stimuli sequences atdifferent luminosities(0,0.3,0.6,1.3and2.0 sintervalwasallowedtoelapsebetweenstimulation wavelengths. A 15–30 sbetweenpresentationsofdifferent interpulse intervaland10–15 s sweredeliveredingroupsofthreewitha2–3 Light pulsesof0.5–1.0 response oftheS-celltoUVRandvisiblelightacrosslevelsluminosity. interneuron. Anadditionalfivepreparationswereusedtoexaminethe more detailbecauseitisaknown(visible)light-responsive,higherorder rs,I,Zcoa,D n or,V. Torre, and D. Zoccolan, I., Arisi, ua .G . oln .adJfry W. H. Jeffrey, and P. Boelen, A.G.J., Buma, A.T. Banaszak, References Funding Competing interests Acknowledgements Data analysis all statisticalanalyseswereperformedwithSPSS(IBM,Armonk,NY, USA). ( individuals wasthenaveragedtoyieldthesummarygraphspresentedhere delivered ateachwavelengthconditionofluminosity. Datafromfive fashion. Allindividualresponsesweretheaveragevaluesfromthreepulses emission) andluminosity(interposedNDfilters)wereanalyzedinasimilar presented totheheadandtailacrosswavelengthrange(colorofLED was definedasP between meanswhensignificantresultswerefound.Statisticalsignificance hoc measures ANOVA, withthetwofactorsbeingwavelengthandtime.A groups ofanimals).Alldatawereanalyzedusingatwo-wayrepeated- each wavelengthacrossfourtimepointsinofthefivesubjects( experiments werebasedontheaveragenumberofvaluesfromthreetrialsat n =5). AllfiguresweregeneratedwithPhotoshopCS3(AdobeSystems)and 138 for zooplanktonverticaldistributioninthe watercolumnoflowDOClakes. Damaging UVradiationandinvertebrate :conflictingselectivepressures Cambridge: TheRoyalSocietyofChemistry. Organisms andEcosystems collective behaviorinmedicinalleeches. aquatic organisms.In activity inthemedicinalleech. Chemistry. Helbling andH.Zagarese),pp.331-358.Cambridge:TheRoyalSociety of whole-body shorteninginthemedicinalleech. aquatic organisms.In test (Fisher’s LSD)wasconductedtoexaminepossibledifferences , 603-612. (2003). Photoprotectivephysiologicalandbiochemicalresponsesof <0.05 foralltests.ThedataonS-cellresponsestolight UV EffectsinAquatic OrganismsandEcosystems UV EffectsinAquaticOrganismsandEcosystems (2011). Statisticalcharacterizationofsocialinteractionsand J. Exp.Biol. (ed. E.W. HelblingandH.Zagarese),pp.23-58. (2003). UVRclimatology. In (2001). Distributedmotorpatternunderlying (1984). A sensorysysteminitiatingswimming J. Neurophysiol. 108 J. Neurophysiol. , 341-355. (2003). UVR-induced damagein Θ 106 ) inthegroupresponse , 78-90. UV EffectsinAquatic 86, 2475-2488. (ed. E.W. (ed. E.W. Oecologia (2004). post n =5 rs,K .adMse K.A. Mesce, and K.M. Crisp, alo,T n cen A. McVean, and T. Carlton, ec,K .adPec-hmmr,J.T. Pierce-Shimomura, and K. A. Mesce, J. Fernandez, W. B. Kristan, and K.A. Mesce, T., Esch, C.R. Fourtner, and C.D. Drewes, Purschke, and D. Arendt, H., Hausen, K., Tessmar-Raible, J., Gosda, C., Döring, W. O. Friesen, and Y. S. Derosa, al .B n rt,S.G. Britt, and J.B. Earl, C.M. Lent, and M.H. Dickinson, ady .adKitn .B,Jr W. B., Kristan, and Q. Gaudry, T. Taxt, and J.K.S. Jansen, A.R., Gardner-Medwin, ree,W. O. Friesen, M.G.F. Fuortes, and R. Fioravanti, iel .M,Sa,B .adKitn .B Jr. W. B. Kristan, and B.K. Shaw, L.M., Misell, Mann, Jr W. B., Kristan, and G.S. Stent, J.R., Kretz, an,F n elgio M. Pellegrino, and F. Magni, C.E. Williamson, and D.M. Leech, et .M n ikno,M.H. Dickinson, and C.M. Lent, ady .adKitn .B,Jr W. B., Kristan, and Q. Gaudry, ad .F,Hrod . i,M .M n i D. Li, and M.L. Lim, J., Horwood, G.V. M.F., Land, Simpson, and S.J. McGirr, Jr, W. B., Kristan, M.P. Nusbaum, and Jr W. B., Kristan, A.G. Dyer, and L. Chittka, P. G., Kevan, J. Viitala, D. and Kueh, H. and J. Siitari, Jellies, E., G.H. Korpimäki, Jacobs, M., Koivula, J., Honkavaara, aasy .adFots M.G.F. Fuortes, and A. Lasansky, ec,D .adJhsn S. Johnsen, and D.M. Leech, M.S. Laverack, aly .M,Cefeo,J n aear D.A. Wagenaar, and J. Cienfuegos, C.M., Harley, B.R. Hargreaves, ele,J,Jhne,K n oasn J. Johansen, and K. Johansen, J., Jellies, Harley Comp. Biochem.Physiol. systems inhostdetectionandlocationthemedicinalleechHirudomedicinalis. medicinal leech. modulation ofdecision-makingneuralpathwaysdescendingfromthebrain making inthemedicinalleech. Gene Expr. Patterns photoreceptor celldifferentiation: insightsintoR7andR8cellsubtypecommitment. Oligochaete. with thescanningelectronmicroscope. Exp. Biol. organization offiberpathways. Chemistry. Helbling andH.Zagarese),pp.293-327.Cambridge:TheRoyalSocietyof Behav. Neurosci. switching: understandinghowlocomotor patternsareturnedonandoff. Embryology ). –astudyintheleech G. and generalizedchordshorteningreflexes intheleech. the medicinalleech. alters thedepthdistributionof of tactilesensoryterminals. leech. the leech. medicinal leech, locomotion andpreysensingintheleech. Res. 351. reflecting scalesofajumpingspider. in themedicinalleech. lessons fromcolourvisioninbeesandbirds. Ultraviolet visionandforaginginterrestrialvertebrates. Zagarese), pp.457-481.Cambridge:RoyalChemistrySociety. vision. InUVEffectsinAquaticOrganismsandEcosystems visual cellsoftheleech, swimming inthemedicinalleech. 143-160. mechanosensory neuroneresponsestoskinstimulationinleeches. leech swimming. leech. early projectionsofindividualperipheralsensoryneuronsintheembryonicmedicinal peripheral circuitlinkingmultipleoscillators. pathways intheleech, Biol. regulated multisensoryintegrationforpreylocalizationinthemedicinalleech. 105. Cambridge:TheRoyalSocietyofChemistry. in AquaticOrganismsandEcosystems (2013). Evolutionofclitellatephaosomesfromrhabdomericphotoreceptorcells .M,Rsi . inugs .adWgna,D. Wagenaar, and J. Cienfuegos, M., Rossi, C.M., , 214 K. H. 90, 13-21. Acta Physiol.Scand. J. Neurobiol. , 3801-3807. 92, 255-275. J. Physiol. Front. Zool. . NewYork, NY: Pergamon Press. (1962). (1992). Ultravioletvisioninvertebrates. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 (1978). Structureoftheleechnervecord:distributionneuronsand Biol. Bull. (1981). Physiologyofwatermotiondetectioninthemedicinalleech. (1969). Mechanoreceptors,photoreceptorsandrapidconduction J. Exp.Biol. (2003). Water columnopticsandpenetrationofUVR.In J. Physiol.(Paris) Hirudo medicinalis 4 , 1-5. L. 25, 1187-1199. ece Hrdna TheirStructure,Physiology, Ecologyand Leeches (Hirudinea) J. Comp.Physiol.A 6 227 J. Comp.Physiol.A , 687-694. 10, 52. J. Comp.Physiol.A Hirudo medicinalis 177 (2012). Centrallypatternedrhythmicactivityintegratedbya Hirudo medicinalis , 173-194. 104 , 363-371. (1993). A comparisonoftheperformancetwosensory Nat. Neurosci. 87, 30A-31A. 209 (1978). Neuralmechanismsunderlyingthesegmental , 273-277. (2006). Expressionof Daphnia J. Neurosci. (2006). Beyondthecentralpatterngenerator:amine J. Comp.Neurol. (2003) Behavioralresponses–UVRavoidanceand (1984). Serotoninintegratesthefeedingbehaviorof (1981). Morphologyofleechsensilla:Observations , 1746-1756. J. Neurosci. (2009). Behavioralchoicebypresynapticinhibition (1989). Hindsightandrapidescapeinafreshwater (1984). Feedingbehaviorofthemedicinalleech, (1969). Thesiteoforiginelectricalresponsesin 78, 743-747. : feedingasadominant behavior. (1972). Analysisofresponsesinvisualcells Proc. Biol.Sci. (2001). InsituexposuretosolarUVradiation (2010). Feeding-mediateddistentioninhibits . Limnol.Oceanogr. (ed. E.W. HelblingandH.Zagarese),pp.61- Biol. Bull. 154 (1982-1983). Thedualroleofserotoninin . J.Exp.Biol. (2001). Limitstothesalienceofultraviolet: 154 J. Exp.Biol. 12, 1450-1457. . J.CellBiol. (1994). Specificpathwayselectionbythe 106 22, 11045-11054. (2002). Evidenceforsequentialdecision J. Comp.Physiol.A (2010). Sharedstrategiesforbehavioral , 457-471. J. Exp.Biol. , 449-455. 30, 9753-9761. , 1-37. (1976). Photosensoryinputpathways 180 (1998). Behavioralhierarchyinthe 160 Am. Zool. 274 , 165-191. (2007). Opticsoftheultraviolet , 383-393. (1973). The“giant”axonofthe Drosophila 216 Oikos 50, 129-140. 42, 241-252. , 1583-1589. J. Comp.Physiol. 204 , 1890-1897. 46, 416-420. (1982). Behaviouraland (2011). Developmentally e.W HelblingandH. (ed. W. , 2571-2580. 98, 505-511. (Annelida, Sedentaria, (2013). Discontinuous 32, 544-554. 198 rhodopsins during , 567-582. J. Exp.Biol. Behav. UV Effects 124 (2002). J. Exp. , 339- Front. 96, J.

The Journal of Experimental Biology eesn E.L. Peterson, E.L. Peterson, D.P. Morris, and C.L. Osburn, eesn E.L. Peterson, eesn E.L. Peterson, D.A. Baylor, and J.G. Nicholls, ul .G n ec,K.A. Mesce, and J.G. Puhl, RESEARCH ARTICLE ulr .J,Ncol,J .adSet G.S. Stent, and J.G. Nicholls, K.J., Muller, aly .L,Mde,B . ols .M n ulr K.J. Muller, and N.M. Boulis, B.K., Modney, C.L., Sahley, ezpuo,S,Sna,P,Dneii,D,Hdr .P n ats R. Santas, and D.-P. Häder, D., Danielidis, P., Santas, S., Reizopoulou, Richa,H.,Kumar, A.,Tyagi, M.B.andSinha,R.P. R.P., Rastogi, hw .K n rsa,W . Jr W. B., Kristan, and B.K. Shaw, acd,E,Zeg . hsr,M,Bg,E .adBit S.G. Britt, and R.T. E. Sawyer, Bagg, M., Phistry, L., Zheng, E., Salcedo, Comp. Physiol.A cells ofthesupraesophagealganglion. sensory neuronsinCNSoftheleech. leech. Royal SocietyofChemistry. Ecosystems organic matterinnaturalwaters.In Spring Harbor, NY: ColdSpringHarborLaboratory. crawling inthemedicinalleech. J. Comp.Physiol. Physiol. A medicinal leech:motorpattern,sensorybasis,andinterneuronalpathways. Neurosci. interneuron essentialforsensitizationandfulldishabituationofleechshortening. Photobiol. effects oninvertebrateanddiatomassemblagesofGreece. 10.4061/2010/592980 mechanisms ofultravioletradiation-induceddamageandrepair. basis forultravioletvisionininvertebrates. Systematics. J. Neurobiol. 14, 6715-6721. 177 56, 172-180. (1986). (ed. E.W. HelblingandH.Zagarese),pp.187-217.Cambridge,The Oxford: ClarendonPress. (1985b). Visual interneuronsintheleechbrainIII.TheunpairedHcell. , 667-681. (1985a). Visual interneuronsintheleechbrainII.Theanteriorvisual (1984b). Two stagesofintegrationinaleechvisualinterneuron. (1984a). Photoreceptorsandvisualinterneuronsinthemedicinal 155 156 15, 413-428. Leech BiologyandBehaviour:FeedingBiology, Ecologyand , 543-557. , 719-727. J. Neurosci. (2008). Dopamineactivatesthemotorpatternfor (2003). Photochemistryofchromophoricdissolved (1968). Specificmodalitiesandreceptivefieldsof (1995). Thewhole-bodyshorteningreflexofthe J. Neurophysiol. J. Comp.Physiol.A UV EffectsinAquaticOrganismsand J. Neurosci. 28, 4192-4200. (1981). Neurobiology oftheLeech 31, 740-756. 23, 10873-10878. 156 (1994). TheScell:an , 707-717. (2003). Molecular (2010). Molecular J. NucleicAcids J. Photochem. (2000). UV J. Comp. . Cold J. J. . oa,M .adDee,C.D. Drewes, M. and Heisenberg, M.J. and Zoran, C. Desplan, S., Yamaguchi, A.L. Willard, idl,M . rnej . tvk,S . kmn,M n adnl,K . III K.S., Macdonald, and M. Nkamany, S.Y., Utevsky, P., Trontelj, M.E., Siddall, Jr W. B., Kristan, and B.K. Shaw, oa,M . rws .D n ornr C.R. Fourtner, and C.D. Drewes, M.J., Zoran, ih,R .adHdr D.-P. Häder, and R.P. Sinha, mt,K .adMcgo E.R. Macagno, and K.C. Smith, az B. Walz, S. Roy, and W. F. Vincent, ul,T n un,W. G. Quinn, and T. Tully, M.J. Tovée, ak .E,Shnlr .W n rs M.T. Arts, and D.W. Schindler, S.E., Tank, swimming bythemedicinalleech. systems. J.Comp.Physiol.A oligochaetes: Variations indesignandfunctionofevolutionarilyconservedgiantfiber Acad. Sci.USA photoreceptor subtypestospectralwavelengthpreferencein Ca accumulation. photoreceptor. I.IntracellulartopographyasrevealedbyOsFeCNstainingandinsitu leeches arenot (2007). Diversemoleculardatademonstratethatcommerciallyavailablemedicinal leech. parallel interneuronalpathwaysinthewhole-bodyshorteningreflexofmedicinal of thetubificidworm, production: damage,protection,andrecovery. Photochem. Photobiol.Sci. paired giantfibersystem. ommatidia. Daphnia magna mutant Drosophilamelanogaster four montanelakes. radiation onbenthiccommunities:epilithicfoodqualityandinvertebrategrowthin distribution andfunction. J. Neurophysiol. (1982). Ca J. Comp.Physiol.A (1981). Effects ofserotoninonthegenerationmotorprogramfor The JournalofExperimentalBiology(2014)doi:10.1242/jeb.094243 (1995). Ultra-violetphotoreceptorsintheanimalkingdom:their Hirudo medicinalis 107 J. CellBiol. 2+- (Crustacea, Branchiopoda).A fourthspectralclassinsingle , 5634-5639. Oikos sequestering smoothendoplasmicreticuluminaninvertebrate Branchiura sowerbyi 82, 1114-1123. Trends Ecol.Evol. J. Comp.Neurol. (1985). Classicalconditioningandretentioninnormal 103 (1993). Solarultraviolet-Bradiationandaquaticprimary 1 , 225-236. (2002). UV-induced DNA damageandrepair:areview. 161 93, 839-848. , 651-667. 166 . J.Comp.Physiol.A (1990). UVphotoreceptorsinthecompoundeyeof , 729-738. J. Neurosci. . Proc.Biol.Sci. , 597-606. (1999). RelativerolesoftheScellnetworkand (1987). Rapidescapereflexesinaquatic : Structuralandfunctionalasymmetryina 275 10, 455-460. (2003). Directandindirecteffects ofUV Environ. Rev. 1 , 76-86. (1988). Thelateralgiantinterneurons , 936-944. 274 157 , 1481-1487. , 263-277. (2010). Contributionof 1 , 1-12. Drosophila . Proc. Natl. 985

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