bacterial-feeding nematodethatformsaspecies-specificandfemale host attractsC.japonica that thepresenceofaspecificvolatilekairomonereleasedby C. japonica of Agriculture, Saga University,of Saga 840-8502,Japan. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3197-3199doi:10.1242/jeb.105353 Received 13March 2014;Accepted3July2014 ‡ 1 EPNs respondtovolatilesfrominsectsandCO infective juveniles(IJs)ofsteinernematidandheterorhabditid been reportedinentomopathogenicnematodes(EPNs). The The recognitionandimportanceofvolatilesinhostsearchinghave for nematodestoorient,particularlythosefromdistantlocations. orientation, arepoorlyunderstood.Volatiles couldbeausefulcue mechanisms ofhostfinding,includingrecognitionand strategies varyamongspeciesorecologicalniches,and Grewal etal.,1993;Shapiro2000).However, host-searching et al.,2009)andchemicalcompounds(PyeBurman,1981; temperature (ByersandPoinar, 1982),electricfields(Shapiro-Ilan of host-relatedstimulisuchasvibrations(Torr etal.,2004), nematodes. Host-searchingnematodesrespondtodifferent types Finding ahostisthemostcriticalstepforparasiticandphoretic Navigation KEY WORDS:Dauer, Kairomone,Nictation,Olfactometer, from threeotherinsectsortoCO significantly attractedtotheairfrom a Y-tube olfactoryassaysysteminwhichC.japonica themselves toahostusingvolatilecuefromthehost,wedeveloped determine whether underlying theestablishmentofthisphoresyremainunknown.To predominantly foundonfemalephoretichosts,butthemechanisms (DL), thenon-feedingandphoreticstageof species-specific phoresywith this behaviourislimited.Caenorhabditisjaponica nematodes; however, informationondirectcuesfromhoststoevoke Host orientationisthemostimportantstepinhost-searching Etsuko Okumura japonica Host orientationusingvolatilesinthephoreticnematode SHORT COMMUNICATION al., 2010;Aliet2011). infection rates(Rasmannetal.,2005;Hiltpold2010;Ali et (HIPVs), whichfacilitateshostdetection,resultinginhigher al., 2012).IJsarealsoattractedtoherbivore-inducedplantvolatiles 1993; CampbellandKaya,1999;Hallemetal.,2011; Dillmanet Oiwake, Sakyo, Kyoto 606-8502,Japan. Agriculture, Kyoto University,*Present address:Graduate School of Kitashirakawa Japan. AgriculturalSciences, Kagoshima University,School of Kagoshima 890-8580, INTRODUCTION ABSTRACT Author ([email protected]) for correspondence Applied BiologicalSciences, Nematology, Faculty of of Department Laboratory Caenorhabditis japonica DL utilizevolatilesforhostrecognitionandorientation C. japonica 1,2, * andToyoshi Yoshiga DL. Kiontke, HironakaandSudhausisa japonensis DL areabletorecognizeandorient 2 . Theseresultsdemonstratedthat P. japonensis 2 The UnitedGraduate C. japonica but nottotheair 2 1,3,‡ (Lewis etal., . Dauerlarvae establishes a DL were , are nematode locomotionandresponse tovolatiles(datanotshown). during hostsearching(Okumura etal.,2013c);thisimproved japonica japonensis vertically because nematode movement.Inaddition, theassayarenawaspositioned their movements;thus,usingwateragarintheassayarenaimproved 1A,B).Unlikearthropods,nematodesrequirehighhumidityfor (Fig. olfactometer sothat of humidityandlocomotionactivity. Thus,wemodifieda research becauseofsometechnicaldifficulties suchasconditioning However, gives thepossibilityofchoosingbetweentwodifferent stimuli. we developedanassaysystemfor host andorientthemselvestothehost.To examinethispossibility, Thus, itispossiblethatC.japonica surface andquicklyorientthemselvesforembarkationonthehost. the soilsurface,DL havetodetectthepresenceofahostonsoil to ahostinsect. olfactometer anddemonstratedtheirrecognitionoforientation on a developmentalanalogueofIJsinEPNs,arepredominantlyfound a developmentallyarrestedandphoreticstageofthenematode Scott (Kiontkeetal.,2002;Yoshiga etal.,2013).Dauerlarvae(DL), host-biased phoresywiththeburrowerbugParastrachiajaponensis one armofthe olfactoryresponses(Smithetal.,1994).Volatiles passthrough Y species-specific phoresyarenotwellunderstood. essential onlyforthenematode,butmechanismsofforming (Okumura etal.,2013b;Yoshiga etal.,2013).Thus,thephoresyis Zuccarini forhernymphs,aswelleggsandnymphalcarcasses the motherinsectstoresfruitsof transport tofoodresources,suchasthenestofhostinsectwhere insect toprolongitsownsurvival(Tanaka etal.,2012)andfor the nematodebenefitsfromthisphoresy. Thenematodeusesthehost attraction isnecessary. studies (Bargmann, 2006).Thus,anewmethodtoevaluatevolatile plate assay, whichiscommonlyusedinnematodechemoattraction japonica but alsonon-volatiles,anditisdifficult todistinguishwhetherC. and attractsDL.However, hexaneextractscontainnotonlyvolatiles species-specific kairomonethatisdirectlyreleasedbythehostinsect (Okumura etal.,2013a).Thesestudiesindicatethepresenceofa extracts containingbodysurfacecomponentsof specifically embarkon RESULTS ANDDISCUSSION -tube olfactometersarecommonlyusedinexperimentsinvolving In theolfactoryassayusing In ourpreviousstudy, wedemonstratedthat Because C.japonica P. japonensis DL areattractedtovolatilesornon-volatilesintheagar DL movedtowardsthedirectionof airflowfrom Y soon aftertheinitiation oftheexperiments, and 43% -tube olfactometersarenotcommonlyusedinnematode Y adult femalesthroughouttheyearinfields.Only -tube andcleanairpassesthroughtheother;this C. japonica C. japonica P. japonensis DL embarkonahostinsectthatwanders DL haveatendencytomoveupward P. japonensis Schoepfia jasminodora C. japonica DL could freelymoveinit DL recognizevolatilesfroma and areattractedtothehexane as thetestinsect, DL usingaY C. japonica P. japonensis Siebold & Y 3197 -tube -tube DL C. P.

The Journal of Experimental Biology study, to attract 3198 yet didnotattract orCO significantly higherwhenP. japonensis observed. Thepercentageofnematodesinthetestareawas used asthetestinsect,andnoattractiontowardtheseinsectswas Thunberg, inoculation areaineachassaywhen more thanhalfoftheinoculatedDL wererecoveredfromthe while only16%(range0–49%)wereinthecontrolarea.Incontrast, (range 13–89%)ofinoculatednematodeswerefoundinthetestarea SHORT COMMUNICATION C IJs ofEPNs(Hallemetal.,2011). Inourstudy, however, CO asaby-productofrespirationattractsDL of of a cueforC.japonica.Theseresultsalsoindicatethatthemovement CO by different typesofvolatilessuchasHIPVs,insectand and caninfectmostinsectorders(Poinar, 1979),andIJsareattracted is notwellunderstood(Strong,2002),EPNshaveabroadhostrange ecological background.AlthoughtheecologyofEPNsinwild utilization ofvolatilescouldvaryamongspecieswiththeir nematodes. specific volatilekairomonethatattractshost-findingstage this isthefirstreportthatdemonstratespresenceofaspecies- specific hostandorienttowardthese.To thebestofourknowledge, demonstrate that evoked byspecificvolatilesproducedthishostinsect.Ourresults Aeration Many nematodesutilizevolatilesforhostsearchingbutthe Jinfectivejuvenile herbivore-inducedplantvolatile generalizedlinearmodel entomopathogenicnematode IJ dauerlarvae HIPVs GLM EPN DL List of abbreviations List of unit C. japonica

% DL 2 100 (Lewis etal.,1993;CampbellandKaya,1999;Hallem 20 40 60 80 P. japonensis A. sordidus, 0

C. japonica Acrida cinerea Water bottle 11 (8) (121) 14.7 26.8 15.8 43.0 2 DL towardstheairfrom was used(Fig. C. japonica,whichsuggeststhatCO * A. sordidus C. japonica A. cinerea Y-tube DL (Fig. Flow meter 17 58 14 11 11 Thunberg orOxyayezoensis A. cinerea and Insect DL recognizevolatilesfromtheir 1C). Thethreeinsectsusedinthis (20) 13 16 1C; 61 12 O. yezoensis,alsoproducedCO O. yezoensis Control bottle P Test bottle P. japonensis <0.001). CO was usedthanwhenother (10) Assay 17 Acanthocoris sordidus 61 13 arena 9

CO (21) 10.5 64.8 11.3 11.3 Inoculation C. elegans 13 B 2

2 appears tobe 2 Shiraki were Test area emitted by may notbe Test Control Inoculation area Downward 2 failed 45 mm and 2 Downward Control biological activitiessuchasCO potential hosts,IJsofEPNsmayutilizesimpleindicators and host-searchingstrategies.To effectively approachavarietyof IJ EPNsand orientation in understand themechanismandevolutionofhostrecognition responses toknownchemicalsarethusnecessarybetter medium (Haraetal.,1981)seededwith City, SagaPrefecture,Japan,wasusedfortheexperiments.Dogfoodagar japonensis Caenorhabditis japonica from phoresy withP. japonensis,andDL areattractedonlybyvolatiles 2011; Dillmanetal.,2012).Incontrast, study oftheJapanesehorntail(MatsumotoandSato,2007).The constructed anolfactometerthatwasamodifiedversionofusedinthe To determinewhethervolatilesfrominsectsattractnematodes,we japonensis orientation in kairomone maybeimportantfactorsforhostrecognitionand activities andincreaseinthesensitivityresponsetoaspecific The decreaseinresponsetosimpleindicatorsfrombiological instead exclusivelydependonaspecificchemicalcuefromthehost. DL maynotutilizethesesimpleindicatorsashoststimuli;they various volatilesasadditionalinformation.Incontrast, Nematodes MATERIALS ANDMETHODS Olfactometry al., 2010).Inbrief,asterileyellow200 Nictating and ChalmersOP50wasusedtoobtainalarge numberofnematodes. at thecentreofa100 Ltd, Tokyo, Japan)wasverticallyplacedsuchthatthetaperedsideup picked upusingafineneedleandusedforsubsequentexperiments. after inoculation,massesofnictatingDL atthepointofyellowtipwere E. coli P. japonensis OP50 andthenematodeswereinoculated.Approximately5 C. japonica inoculation point.(C)Olfactoryresponseof small circleintheinoculationareaindicatesnematode control, inoculationanddownwardareaswasdetermined.The the startofexperiment,numbernematodesintest, (B) Enlargementoftheassayarena.Approximately10 inoculation areawhenotherinsects orCO In contrast,morethanhalfoftheinoculated DL stayed withinthe of nematodesmovedtowardsvolatilesemittedby Acrida cinerea and tofourinsectspecies: Fig. using ageneralizedlinearmodel(GLM). * was noattractiontootherinsectsor CO not movetoward When theassayarenawassethorizontally, thenematodesdid inoculation area,andtheassayarenawassetvertically(B). 30–100 dauerlarvae(DL)wereinoculatedatthecentreof drifted theinoculatednematodesdownward.Approximately glass tube(innerdiameter, 12 pump, awaterbottlecontainingdeionizedwater, aY-shaped larvae. adjusted to0.4 and anassayarena(Y-shaped glasstube).Airflowratewas collected fromHinokumaMountainPrefecturalPark,Kanzaki The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105353 that attract C. japonica

C. japonica.Analysisofthevolatilecomponents .Olfactoryassayusing 1. C. japonica. (A) Olfactometer. Theolfactometerconsistsofanair . Thedifferences inresponsetovolatilesbetween

DL werecollectedasdescribedpreviously(Tanaka et ml culturebottleofdogfoodagarmediuminwhich

and Oxyayezoensis l

strain H1isolatedfromanadultfemaleof min C. japonica DL mayreflectthevariationsinhostrange P. japonensis − 1 using aflowmeter. A fasterairflowrate 2 P. japonensis μl pipettip(Watson, FukaekaseiCo., as basichoststimulialongwith Escherichia coli

mm), twoflowmeters,bottles Caenorhabditis japonica (data notshown). DL andstudiesonsensory C. japonica . A significantlyhighernumber 2 , Acanthocorissordidus . Datawereanalysed P 2 <0.001. C. japonica was used,andthere (Migula) Castellani forms aspecific P. japonensis C. japonica

min after DL toCO dauer

days . P. , P. 2

The Journal of Experimental Biology effect ofCO control, inoculationanddownwardareas;Fig. aeration, wedisconnectedthearena,andnematodesineacharea(test, vertically, andaerationwasstarted.Approximately10 water agaratthecentreofinoculationarea(Fig. of thenematodesuspensioncontaining30–100DL wasplacedontothe aeration unit,waterbottles,flowmetersand 1.5% wateragar. Siliconetubeswereusedforallconnectionsbetweenthe adjusted toarateof0.4 Orthoptera arena (thesecond The airpassedthroughthetestandcontrolbottlesconnectedtoassay test bottlecontainedainsect,whereasthecontrolhadnoinsects. Japan), whichwasconnectedtoa100 and 21,respectively. agan C.I. L. Bargmann, Stelinski, and H.T. Alborn, J.G., Ali, sordidus, 16, 2,1and1,respectively. Thenumberofreplicates P. japonensis, 1.Thenumberofinsectindividuals olfactometry assayarelistedinTable was adjustedtoarateof0.4 meters, andtheotherflowmeterwasconnectedtoaerationunit.Air SHORT COMMUNICATION commercial, ornot-for-profitsectors. This researchreceivednospecificgrantfromanyfundingagencyinthepublic, revising ofthearticle. contributed totheconceptionanddesignofexperiments,drafting and E.O. performedtheexperimentsanddataanalyses,draftedarticle.T.Y. The authorsdeclarenocompetingfinancialinterests. for hisadviceondataanalysis. We thankDrS.Tojo forhisvaluablecommentsonthisstudyandDrA.Yamawo 4.54; AbacusConcepts,Inc.,NJ,USA). using ageneralizedlinearmodelwithGaussiandistribution(StatView Ver. Percentages ofnematodesinthetestareawerecomparedamonginsects joint ( humidity, andthendividedintotwolinesbythefirstglass water ina300 air flowrateof0.8–1.0 unit (MAU-2,Tokyo RikakikaiCo.,Ltd,Tokyo, Japan)wassettoobtainan olfactometer usedinthecurrentstudyisillustratedFig. Insects Table l,J . lon .T n tlnk,L. Stelinski, and H.T. Alborn, J.G., Ali, References Funding Author contributions Competing interests Acknowledgements Statistical analysis nematodes. subterranean plantvolatilesattractboth entomopathogenicandplantparasitic elegans ResearchCommunity),pp.1-29. Available at:http://www.wormbook.org. entomopathogenic nematodes. volatiles releasedbycitrusrootsuponfeeding Acrida cinerea Parastrachia japonensis Acanthocaris sordidus Oxya yezoensis

Y 1. Listofinsectsusedinthisstudy -tube: innerdiameter, 12 A. cinerea 2 J. Ecol. , abottleofcompressedCO A. cinerea

ml glassbottletoremoveodoursandmaintainahigh (2006). ChemosensationinC.elegans. In (unsexed adult) usxdaut ThecampusofSaga University, Japan,2010 (unsexed adult) Y 99, 26-35. , -tube), whoselongitudinalhalfvolumewasfilledwith O. yezoensis

l l

(nymph) min min , (adult female) O. yezoensis

− l −

1 J. Chem.Ecol. 1 min . Airwaspassedthrough~150 using aflowmeter(RK200V, Kofloc,Kyoto,

mm; Sansyo,Tokyo, Japan).Airflowwas − 1 and CO using aflowmeter. Insectsusedforthe

ml Pyrextestorcontrolbottle.The and (2010). Subterraneanherbivore-induced 2 2 was connectedtooneoftheflow 36, 361-368. experiments was121,8,20,10 A. sordidus Y (2011). Constitutiveandinduced

-tubes. Approximately20 1) werecounted.To testthe ipee abbreviatus Diaprepes

1B), thearenawasset The campusofSagaUniversity, Japan, 2010 Hinokuma MountainPrefecturalPark,Japan,2007–2009 The campusofSagaUniversity, Japan, 2010 Sampled sitesandyears WormBook used forassaywas

min afterstarting

P. japonensis 1A. Theaeration Y ml ofdistilled -shaped tube (ed. TheC. recruit , μl A. aaa . kmr,E,Knai .adYsia T. Yoshiga, and N. Kanzaki, E., Okumura, R., Tanaka, hpr,D . ei,E . aaaia,S n co,C.W. McCoy, and S. Paramasivam, E.E., Lewis, D.I., Shapiro, yr,J .adPia,G . Jr G.O., Poinar, and J.A. Byers, or . eiae .adWlo,M. J. Wilson, and S. Heritage, P., Torr, T. Yoshiga, and E. Okumura, R., Tanaka, D.R. Strong, D. Kim-Shapiro, and J.M. Elkon, E.E., Lewis, J.F., Campbell, D.I., Shapiro-Ilan, ohg,T,Ihkw,Y,Tnk,R,Hrnk,M n kmr,E. Okumura, and M. Hironaka, R., Tanaka, Y., Ishikawa, T., Yoshiga, Smith, C.M.,Khan,Z.R.,andPathak,M.D. ila,A . ulemn .L,Le .H,Km . trbr,P .adHallem, and P. W. Sternberg, B., Kim, J.H., Lee, M.L., Guillermin, A.R., Dillman, H.K. Kaya, and J.F. Campbell, itod . aoi . ope,S,Khmn,U n ulns T. C. Turlings, and U. Kuhlmann, S., Toepfer, M., Baroni, I., Hiltpold, H.K. Kaya, and J.E. Lindegren, S.F. A.H., DeMarco, Hara, J.M., Yano, Y., Zhang, A.V., Hong, A.R., Dillman, E.A., Hallem, ink,K,Hrnk,M n uhu,W. Sudhaus, and M. Hironaka, K., Kiontke, S. Selvan, and R. Gaugler, P. S., Grewal, asmt,T n ao S. Sato, and R. T. Matsumoto, Harrison, and R. Gaugler, E.E., Lewis, y,A .adBra,M. Burman, and A.E. Pye, kmr,E,Ihkw,Y,Tnk,R n ohg,T. Yoshiga, and R. Tanaka, Y., Ishikawa, E., Okumura, T. Yoshiga, and R. Tanaka, E., Okumura, Rasmann, Jr G.O., T.Poinar, Yoshiga, and R. Tanaka, E., Okumura, system foramodelorganism,C.elegans dauer larvainthenematode 40, 7-12. phoretically activedauerlarvaeof partitioning in location byparasiticnematodes. T. C. Turlings, and nematodes byinsect-damagedmaizeroots. J. Gershenzon, Neoaplectana carpocapsae Caenorhabditis japonica Species-specific andfemalehost-biased ectophoresyintheroundworm CABI Publishing. Entomopathogenic Nematology 147-190. BocaRaton,FL:CRCPress. allelochemicals. In electrical current. B. nitrogen onattraction/repulsion. Proc. Natl.Acad.Sci.USA E. A. Nature volatile rootsignalhelpstocontrolamajorpest. Selection ofentomopathogenicnematodesforenhancedresponsivenesstoa USDA. (AdvancesinAgriculturalTechnology. AAT-W, 16). Agar Medium the EntomogenousNematode,NeoaplectanaCarpocapsaeWeiser, onDogFood/ P. W. nematodes. Sternberg, and Chem. Ecol. entomopathogenic nematodes:behavioralresponsetocontactwithhostfeces. Parastrachia japonensis japonica Forest Society the JapanesehorntailUrocerusjaponicas Can. J.Zool. ambusher entomopathogenicnematodes(Steinernematidae)tohostvolatilecues. accumulations onchemicalandbacterialgradients. CRC Press. Caenorhabditis japonica Zoolog. Sci. Caenorhabditis japonica 216, 568-572. carrier insectbydauerlarvaeofthenematode (2009). Directionalmovementofsteinernematidnematodesinresponseto (2012). Olfactionshapeshost-parasiteinteractionsinparasiticnematodes. 397 . öle,T . eehrt . itod . ope,S,Khmn,U., Kuhlmann, S., Toepfer, I., Hiltpold, J., Degenhardt, T. G., Köllner, S., n. sp.(Nematoda:Rhabditida)associatedwiththeburrowerbug , 485-486. Curr. Biol. 30, 174-177. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105353 19, 1219-1231. (2002). Populationsofentomopathogenicnematodesinfoodwebs.In 71, 765-769. 89, 135-137.(JapanesewithEnglishsummary). (1979). eeohbii bacteriophora Heterorhabditis J. Invertebr. Pathol. Techniques forEvaluatingInsectResistanceinCropPlants 21, 377-383. Nematodes forBiologicalControlofInsects . Naturwissenschaften (: Cydnidae)inJapan. dauer larvae. in thenestofitscarrierinsect, (2011). A sensorycodeforhostseekinginparasitic 109 , inresponsetotemperature. (2007). Anolfactometertestingtheolfactoryresponseof , E2324-E2333. J. Invertebr. Pathol. Caenorhabditis japonica (1999). Howandwhyaparasiticnematodejumps. Int. J.Parasitol. (ed. R.Gaugler),pp.225-240.Wallingford, UK: (1981). (1982). Locationofinsecthostsbythenematode Caenorhabditis japonica 100 J. Exp.Biol. (2004). Vibrations asanovelsignalforhost (2005). Recruitmentofentomopathogenic Exp.Gerontol. . (2013a). Species-specificrecognitionofthe , 134-137. (2013c). Negativegravitacticbehaviorof Neoaplectana carpocapsae Nature to volatiles. (1981). (2002). Descriptionof (2010). A simplemethod to collect -infected hostsandtheeffects of 100 Caenorhabditis japonica (1994). Evaluationofvolatileplant (1993). Responseofcruiserand 34, 997-999. , 205-208. 216 Exp. Parasitol. 434 76, 43-48. J. Exp.Biol. (1993). Hostrecognitionby Monoxenic MassProductionof , 1470-1474. , 732-737. (2012). Lowsurvivorshipof Behaviour 47, 388-393. , apotentialcomparative Journal oftheJapanese . Nematology Parastrachia japonensis. (2013b). Propagationof Nematological Research 213 . BocaRaton,FL: 51, 13-20. 79, 1-10. (2000). Nitrogen , 2417-2423. Caenorhabditis Oakland, CA: . 4 : nematode J. Exp.Biol. , 933-941. (2013). (2010). 3199 , pp. J.

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