Author(s) 神田, 侑奈; 並川, 寛司; 渡部, 英昭
Citation 北海道教育大学紀要. 自然科学編, 70(1): 1‑15
Issue Date 2019‑08
URL http://s‑ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/10518
Rights
BiologyoftheSpotted-WingDrosophilasuzukii(Diptera:Drosophilidae)in Hokkaido,NorthernJapan.II.SeasonalFluctuation
KANDAYuna＊,NAMIKAWAKanji＊＊andWATABEHideaki＊＊＊
＊，＊＊BiologicalLaboratory,SapporoCampus,HokkaidoUniversityofEducation,Sapporo002-8502
＊＊＊HokkaidoUniversityMuseum,HokkaidoUniversity,Sapporo,060-0810
北海道におけるオウトウショウジョウバエの生態．Ⅱ．季節消長
神田 侑奈＊・並川 寛司＊＊・渡部 英昭＊＊＊
＊，＊＊北海道教育大学札幌校生物学教室
＊＊＊北海道大学総合博物館
ABSTRACT
Seasonalfluctuations,photoperiodicresponses,andcold-hardinessofthespotted-wing Drosophila(Sophophora)suzukiiwerestudiedinHokkaido,northernJapan.Thespecies appearedinearlyJuly,rapidlyincreaseditspopulationsize,andcontinuedactivelybreeding untilmid-October.Thespeciesrepeatedmainlythree,andinoccasionsfour,generations duringaperiodfromJulytoOctober.Thedevelopmentoffemale’sovarieswasdelayed undershortdaylengthsat15°C,i.e.,approximately80%ofthe8-day-oldfemalesremained intheundevelopedovarystageinLD10:14(10hoursoflight:14hoursofdark),whereas approximately20%didsoinLD16:8andLL(continuouslight).However,thesuppression ofovariandevelopmentdidnotlastlong,andmostofthe16-day-oldfemalesmaturedtheir ovaries.Dorosophilasuzukiiadultsthathadbeenrearedundershortdaylengthsfromthe imaginalstageat15°Cweremoretoleranttolowfreezingtemperaturesthanthosereared underlongdaylengthat18°C;abouthalfoftheadultscouldnotsurviveat-4°Cto-6°Cfor 24hoursevenintheformerregime.TheweakresponseofD.suzukiitoshortphotoperiods andtoweakcold-hardinesswasnotadaptiveanddidnotpermitthemtosurvivethesevere winterofHokkaido.Thismighthaveresultedintheabsenceofitsadultsinthenextspring.
ComparedtothephenologyofD. suzukiisurveyedinthe1970s,itappearedaboutamonth earlierinSapporo.
Keywords:Seasonalactivity,generationalternation,photoperiodicresponses,cold-hardiness,overwintering
＊Presentaffiliation:Esashi-kitaJuniorHighSchool;＊＊＊Correspondingauthor:e-mail:[email protected]
１．INTRODUCTION
Thespotted-wingDrosophilasuzukiiisthefirstnewspeciesdescribedbyMatsumura(1931)inthe drosophilidfaunaofJapan.AsitsJapanesename“Ōtō-shōjōbae(Ōtō=cherry,shōjōbae=vinegarfly)”
suggests,thespeciesisaseriouspestinsectforfruitssuchascherry,peaches,andgrapes(Kanzawa, 1939).DrosophilasuzukiiisessentiallyanOrientalmemberinthedrosophilidbiogeography,occurringin northernIndia,SoutheastAsia,southernandeasternChina,Korea,andJapan(Okada,1988).Outsideits originalrangeofdistribution,itsoccurrencewasfirstrecordedinOahu,Hawaiiin1980(Kaneshiro,1983).
Then,theflywasdiscoveredinNorthAmericaandEuropein2008,andithasbeenrecentlyreportedin manydistrictsofNorthAmerica,SouthAmerica,Europe,andtheMiddleEast(Hauser,2011;Calabriaet al.,2012;Cinietal.,2012;VilelaandMori,2014;Orhanetal.,2016;Lavrinienkoetal.,2017).
WehavestudiedmanykindsoffeedingandbreedingsubstratesutilizedbyD.suzukiiinHokkaido, northernJapan,anditsbreedingrecordsshiftedexclusivelyinthelatesummerandautumn(Kandaet al.,2019).Hokkaidoisinthecooltemperatezonewithaharshclimateandheavysnowinthewinter.
MostspeciesofwilddrosophilidsnativetoHokkaidospendthewinterinastateofreproductivediapause inducedbyshortdaylengths.TheyappearfromlateApriltoearlyMay,afterthesnowmelts,alternate inrepeatgenerationsseveraltimesuntilmid-SeptemberandthenenterhibernationinOctober(Toda andKimura,1978;Watabe,1979;IchijoandBeppu,1990).Ontheotherhand,D.suzukiiisabsentfrom ApriltoearlyJulyinmostareasofHokkaido,anditisconsideredthatthespeciesisunabletosurvive the winter outdoors (Momma. 1957; Wakahama, 1957). Thus, we studied seasonal fluctuation, photoperiodicresponse,andcold-hardinessofD. suzukii usingfieldsurveysandlaboratoryexperiments.
２．FIELD SURVEY AND LABORATORY EXPERIMENTS
Seasonal fluctuations:Periodicalcollectionswerecarriedoutduringtheactiveseasonsusing“retainer- type”trapsbaitedwithfermentedbananas(Toda,1977).SixtrapsweresetupfromApriltoNovember duringfivesuccessiveyears(1975to1979)attheBotanicalGardenofHokkaidoUniversity(UBG),in thecenterofSapporocity(43°N,141°E).Trapcollectionsweremadewithtwotrapsin2016and2017at thecampusofHokkaidoUniversityofEducation(CHU)inthenorthernsuburbsofSapporo,beingabout 15kmfromthecenterofSapporo.FlysampleswerefixedwithKahle’ssolutionor70%ethanoland removedtothelaboratoryinweeklyintervalswiththerenewalofbananabaits.TheovariesofalltheD.
suzukiifemaleswereexaminedtotraceseasonalchangesinreproductiveconditionsunderabinocular stereoscopicmicroscope(OlympusSZ-PT).Thereproductiveconditionofthefemale’sovarieswas classifiedintofourstages:StageI,undeveloped(beforeyolkformation);StageII,developing(yolkis accumulatinginoocyte);StageIII,mature(completelyformedeggswithfilaments);StageIV,post- matureordegenerated(WatabeandBeppu,1977).
Annual fluctuations of natural populations:Wemadethetrapcollectionsfortwoweeksinearly Octoberin25successiveyears(1993to2018)inCHU,followingthesamemethoddescribedabove.Based onthedata,weevaluatedacorrelationbetweensamplesizeandmeteorologicaldata(monthlymean temperatureandmonthlyprecipitation)fromApriltoOctoberineachofthoseyears,usingsimpleand
multipleregressionanalyses,withtheRsoftware(RcoreTeam,2016).Weadoptedthemeteorological dataofSapporo(studyarea),Hakodate(thesouthernmostcityofHokkaido),Sendai(facingtothe PacificcoastintheTohokudistrict),Yamagata(facingtotheJapanSeaandfamousforcherryproducts inTohoku),andHachi-Ohji(Kantodistrict)nearMusashi-Murayamawherethewintercollectionwas made.ThelocalitiesareshowninFig.1.
Photoperiodic responses: SeveralfemalesofD.suzukiiwerecollectedatCHU,toestablishliving strains.AdultfemaleswereallowedtoovipositonusualDrosophilamedium(dryyeast25g,sucrose25g,
Figure 1. MapshowingcollectionlocalitiesofexperimentalstrainsofDrosophila suzukii(①,③,⑦,⑪,⑭)and itssiblingspeuesD. subpulchrella(⑨,⑪,⑬),andthemeteorologicalstations(①,②,④,⑤,⑥)used foranalysesofcorrelationbetweenannualfluctuationsofD. suzukii andclimaticdata.Drosophila suzukiiwasreportedtobeunabletooverwinteroutdoorsintwolocalitieswiththeopencirclesof Sapporo (①) and Mt. Dakesan (⑫), whereas it was collected during the winter months from DecembertoFebruaryinthreelocalitieswiththeclosedcirclesofMusahi-Murayama(⑥),Kariya(⑧) andTakamatsu(⑩).Theshadedareainthemapshowsthedistributionrangeofevergreenforestsof Castanopsis sieboldii andC. cuspidate(cf.Kamijo,2009).Localities:①Sapporo,②Hakodate,③Morioka,
④Yamagata,⑤Sendai,⑥HachiojinearMusashi-Murayama,⑦Hachijo-Island,⑧Kariya,⑨Okazaki,
⑩Takamatsu,⑪Matsuyama,⑫Mt.Dakesan,⑬Yakushima-Island,⑭Shanghai.
malt25g,cornmeal25g,agar4gandpropionicacid2.5mLin500mLofwater).Theywereplacedina glassvial(30mmindiameter:180mminlength)withfood,andkeptat18°CinaSANYOIncubator MIR-153.Forexperiments,flies weretransferredtovariousphotoperiodicconditionsat15°C,18°C,and 23°Cfromtheeggstage,invariouslight:dark(LD)cycles:LD10:14,LD11:13,LD12:12,LD16:8,and continuouslight(LL).Newlyemergedadultswerecollectedeveryday,transferredtoanewvialwith freshfood,andculturedunderthesameconditionsasthepre-imaginalstages.Thedevelopmentalstatus ofthefemale’sovarieswasexaminedinRingersolutionbydissectionunderastereoscopicmicroscope.
Femaleswithundevelopedovarieswereassumedtobeindiapause.Reproductivediapausewasalso characterizedinvirgins,andthusfemale’sspermathecaewereobservedforspermsunderthemicroscope (OlympusCX-41).Inmostcases,approximately30to50femaleswereexaminedateachobservation point.InadditiontotheSapporostrainofD. suzukii,fourgeographicstrainswerestudiedfortheir photoperiodicresponses,includingtheMorioka(40°N,141°E)inTohoku,Hachijo-Island(33°N,140°E) inKanto,Matsuyama(34°N,133°E)inShikokuandShanghai(30°N,122°E)strainsinChina(Figure1).
ThesegeographicalstrainsweremaintainedattheTokyoMetropolitanUniversity.BothMatsuyamaand Shanghaistrainswereiso-femalelinesderivedfromasinglewild-caughtfemale,andtheotherswere establishedbyseveralfemalescollectedinthefields.Further,photoperiodicresponsewasstudiedin threegeographicalstrainsofDrosophila subpulchrella, asiblingspeciesofD. suzukii.Theywerealliso- femalestrains,collectedinYakushimaIsland(30°N,131°E),MatsuyamaandOkazaki(35°N,137°E).
Cold-hardiness:AdultfliesofD. suzukii,thatwereculturedunderashort(LD10:14)andalong daylengths(LD16:8)at15°Cand18°Cfromtheeggstage,weremaintainedunderthesameconditions for8daysaftereclosion.Then,theyweretransferredto5°Cforacclimation,maintainedfor5days,and exposedtolowtemperaturesfrom-2°Cto-6°Cfordifferentperiods.Aftercoldtreatments,adultflies wereplacedatroomtemperature,toallowthemtorecover(cf.HiguchiandKimura,1985）．
３．RESULTS AND DISCUSSION
Seasonal and annual fluctuations: Figure2showtheseasonalchangesinthefemalereproductive conditionsandnumberofmaleindividualscollectedinboththeUniversityofBotanicalGardenin1977 (UBG-77)andinthecampusofHokkaidoUniversityofEducationin2016(CHU-16).InCHU-16,atotal of12,907drosophilidsbelonging2subfamilies,5generaand32specieswerecollected.Ofthem,D.suzukii wasthemostabundant,composingabout30%(n=3616)ofthetotalsamples.InUBG-77,D. suzukii was alsothedominantspecies,with3616individuals.
InCHU-16,averysmallnumberofadultflieswerecollectedfromearlyJulytoearlyAugust.These adultscouldberegardedasthefounders(G0)oftheSapporopopulationofD.suzukii.Duringthisperiod, mostoftheD. suzukii femaleshadmatureovarieswithmanyeggsandactivelyproducedoffspring.The newlyemergedfemaleswithStageIovariesappearedfrommid-Augustandconstitutedalargesummer populationbylateAugust.Theseadultswerethefirstgeneration(G1)derivedfromthefounders.The G1femalesrapidlydevelopedtheirovariesandperformedasecondbreedinginlateAugusttoearly September.Thenewlyeclosedadultsappearedonceagainbymid-September.ThesefliesweretheG2 adultsandtheyperformedathirdbreedingfrommid-Septembertomid-October.TheG3fliesappeared
byOctober,andmostofthefemalesdidnotdeveloptheirovaries,probablyowingtoadecreasein temperaturesorashortageoffoodresourcesbylateOctober.Fromthisfigure,D.suzukiiwasconsidered tohavethree,partiallyfour,generationsperyearinSapporo,althoughthegenerationsmightoverlap fromlateAugusttoearlyOctober.Similarly,inCHU-17,D. suzukii adultsappearedfromearlyJulyand repeatedseveraltimestheirgenerationalternationuntilmid-October.However,thespeciesdidnot constitutealargesummerpopulationinCHU-17.
InUBG-77,itwasdifficulttotracepopulationfluctuationsofD. suzukii,inlargepartduetoimmature females;generationalternationoccurredatleasttwice,inlateAugustandlateSeptember.InUBG-75to UBG-79,D. suzukii appearedinearlyAugust,produceditsprogeny,andincreasedinnumberuntillate October. Unlike 2016, the species did not constitute a large summer population from 1975 to 1979.
Comparingthetwofigures,D. suzukii currentlyappearsinSapporoonemonthearlierthaninthe1970’s.
MostspeciesofwilddrosophilidsappearinlateAprilinHokkaido(WatabeandBeppu,1977;Todaand Kimura,1978;Watabe,1979;IchijoandBeppu,1990;Izumitanietal.,2017,etc.).Ontheotherhand,D.
suzukiiappearsinearlysummer,likedomesticspeciesofD. melanogasterandD.immigrans(Watabe, 1979),andvisitstrapsuntilmid-Novemberwhenwehavethefirstsnowfall.Thewildspeciesalmost ceasebreedinguntilmid-Septemberandenterreproductivediapauseforhibernation(Watabe,1979;
Ichijoetal.,1980;IchijoandBeppu,1990).Drosophila suzukii adults continuetobreeduntilmid-October, andmostofitslarvaeorpupaemightnotdevelopinNovember.
FieldcollectionsofdrosophilidfliesinHokkaidostartedfromthe1950s,andtheresultswerereported in several studies entitled “Drosophila Survey of Hokkaido (DSH)” (Momma, 1957; Takada, 1960;
ToyofukuandKimura,1960;Wakahamaetal.,1963;TakadaandMaekawa,1984,etc.).Thecollection localitiesofD. suzukiiwerecompiledseparatelybymonth,basedonthedatapublishedinDSH(Fig.3).
Figure 2. SeasonalchangesinthefemaleovarianstagesandmaleindividualnumbersofDrosophilasuzukiiin UBG-77(left)andCHU-16(right).ThearrowsinCHU-16showtheseasonalbreedingrecordsofD.
suzukii.
Drosophila suzukiiwasnotfoundinHokkaidoinJune,butitappearedinsouthernHokkaidobyJuly.In August,itsoccurrencewasrecordedinallareasofHokkaido,includingadjacentislands.InSeptember andOctober,D. suzukii wasstillpresentinallareas.Thesecollectiondataimplythatearlysummer populationsofD. suzukiimighthavecomefromthesouth,probablyHonshu.
InCHU-17,atotalof1370adultswerecollectedandthisnumberwasabout10%ofthesamples capturedinthepreviousyear.Suchalargedifferenceinthenumberofcollectedindividualshasoften beenfoundinUBG(Momma,1965).Inthetwo-week-collectionfrom1993to2018,themaximumnumber ofD. suzukiiwas4852in1994anditsminimumoneonly42in2017(Fig.4A).Weanalyzedthedatato determine if there was a correlation between sample size and the monthly mean temperature or precipitation,butwedidnotfindanycorrelationsusingsimpleregressionanalysis.Furthermore,a multipleregressionanalysiswithtemperatureandprecipitationasvariablesdidnotyieldanymeaningful relationship.Figure4BshowstherelationshipbetweenthenumberoftheD. suzukii adultsandthemean temperatureofSeptemberinSapporo.Forthisanalysis,wedividedtheannualcollectiondatainto2 groups,1)theyearswithahighnumberofadultscollected(tentativelymorethan300)and2)those withfewadults.ApositivecorrelationbetweenthemonthlymeantemperatureofSeptemberandthe samplesizewasseenintheformer,withacorrelationcoefficientvalueof0.83(P=0.01);thiscorrelation wasnotfoundforthelatter(r=0.33,P=0.269).TheD. suzukiiadultstrappedinearlyOctoberspenttheir pre-imaginalstagesinSeptember,andthusitisreasonabletoassumethatthetemperatureofSeptember mightaffectthesamplesizeofD. suzukiiadultscaughtinearlyOctober.Itiscertainthatfruitionofwild andcultivatedplantsalsohastheeffectonnaturalpopulationsofD. suzukii.
Photoperiodic responses: IntheSapporostrainat23°C,morethan90%ofthe4-day-oldfemaleshad matureovariesinbothLD10:14andLD16:8.At18°C,aslightdelayonovarianmaturationwasdetected Figure 3. MonthlyoccurrencesofDrosophila suzukiireportedintheseriouspapersof“DrosophilaSurveyof
Hokkaido”.
inLD10:14(Table1).About70%ofthe4-day-oldfemaleshadmatureovariesinLD16:8,whereasabout 55%ofthemdidsoinLD10:14.Suchsuppressionontheovarianmaturationwasnotobservedinother geographicstrainsofD. suzukii, andallfemalesperformedovarianmaturationinbothshortandlong daylengtswithin8daysaftereclosion(Table1).
Figure5AshowsthepercentagesofthefemaleswithundevelopedovariesintheSapporostrain reared, under different photoperiodic conditions at 15°C. About 20% of the 8-day-old females had undevelopedovariesinLD12:12,16:8andLL,whereasabout80%ofthemhadsoinLD10:14;the percentageofundevelopedovariesinLD11:13fellbetweenLD10:14andLD12:12.Table2showsthe percentageofvirginfemalesrearedinLD10:14andLD16:8at15°Cand18°C.Theratioofvirginswas higherintheformerphotoperiodicregimethanthatinthelatterat15°C.Theseresultsindicatethata reproductivediapausewasinducedbyashortphotoperiodat15°C.ThereproductivearrestofD. suzukii didnotlastlongevenat15°Candmostofthe16-day-oldfemalesdevelopedtheirovaries.Figure5B showsthepercentagesoffemaleswithundevelopedovaries,rearedfromLD10:14toLL,8daysafter eclosionat15°C.Thecriticaldaylengthwhenhalfofthepopulationsenteredindiapausewasestimated tobe11.2hoursoflight.TheSapporostrainofD. suzukii recognizesLD12:12tobealongdaylength.It Figure 4. A:AnnualfluctuationsofnaturalpopulationsofDrosophila suzukiifrom1993to2018.Flieswere
collectedbythesamemethodexplainedinthetext,duringtwoweeksinearlyOctober,atCHUin Sapporo.ThemeantemperaturesarethoseofSeptemberinSapporo.B:Correlationbetweenthelarger individualnumbers(morethan300)ofD.suzukiiandthemeantemperatures,basedonthedata showninA.
Table 1. PercentagesoftheDrosophila suzukiifemaleswithmatureovariesrearedundershortandlong daylengthsat18℃ .
Femaleage Photoperiod Sapporo Morioka HachijoIsland Matsuyama Shanghai 4-day-old LD10:14 54.7(75)1) 57.1(56) 83.9(31) 98.4(61) 100.0(44) LD16:8 68.6(51) 53.8(52) 97.3(37) 92.3(39) 97.8(46) 8-day-old LD10:14 82.6(92) 79.4(34) 92.3(52) 100.0(47) 100.0(52) LD16:8 91.7(60) 92.9(42) 100.0(48) 100.0(41) 97.2(72)
1)Thenumberoffemalesexamined.
isoneofthereasonsthatthespeciescontinuesreproductionuntilmid-October,becausealightperiodof 11.2hoursisrecordedduringmid-OctoberinSapporo.MostofthewilddrosophilidsinhabitingHokkaido enterreproductivediapausearoundtheautumnalequinoxdayinmid-September,with12hoursof daytime(cf.TakadaandToyofuku,1960;Minami,1979;Minamietal.,1979;Iwaoetal.,1980;Minamiand Kimura,1980).InthegeographicstrainsfromIowaorMichigan,regionswithnearlythesamenorthern latitudesasHokkaido,aphotoperiodofLD12:12isconsideredasashortdaylengthforD. suzukii(cf.
Stephenetal.,2015:WallingfordandGregory,2016).Thismaybeduetoageographicvariationinthe photoperiodicresponse,becausewealsodetecteddifferentdiapauseincidenceamongJapanesestrainsof thespecies(Fig.6).IntheMoriokaandHachijo-Islandstrains,theratioofundevelopedovarieswas higherinLD:10:14thanthatinLD16:8at15°C,whereasintheMatsuyamaandShanghaistrainsitwas nearlyidenticalbetweenshortandlongdaylengths(Fig.6).
Figure 5. A:PercentagesoftheDrosophilasuzukiifemaleswithundevelopedovaries,rearedundervarious photoperiodicconditionsat15°C.B:Thephotoperiodicresponsecurveattheageof8dayafterthe adulteclosion.Arrowshowsthecriticaldaylengthwhenhalfofthefemalesremainindiapause.
Table 2. PercentagesofthevirginsfemalesofDrosophila suzukiirearedundershortandlongdaylengthsat15 and18℃.
Femaleage Temperature Photoperiod Sapporo Morioka Hachijo
Island Matsuyama Shanghai 4-day-old 15℃ LD10:14 100.0(56)1) ― 94.6(74) 64.2(53) 82.3(62) LD16:8 89.6(48) ― 45.2(42) 33.3(57) 62.4(125) 18℃ LD10:14 38.7(75) 28.6(28) 6.5(31) 6.6(61) 6.8(44)
LD16:8 35.3(51) 7.7(26) 0.0(37) 5.1(39) 0.0(46) 8-day-old 15℃ LD10:14 79.2(72) 84.4(32) 65.8(62) 37.2(43) 9.4(85) LD16:8 64.8(54) 46.7(30) 16.2(37) 9.5(42) 15.2(66) 18℃ LD10:14 6.5(92) 20.6(34) 0.0(52) 0.0(47) 0.0(52) LD16:8 6.7(60) 6.5(31) 4.2(48) 0.(41) 0.0(72)
1)Thenumberoffemalesexamined.
Amongdrosophilidflies,domesticspecieswithtropical-andsub-tropicalorigins,e.g.,D. melanogaster, D. simulnas, D. busckii,and D. immigrans,donotshowanyofdiapausecharacteristics,andtheydevelop theirovariesunderallphotoperiodicregimes(Lumme,1978;LummeandLakovaara,1982;Watabe,1979).
ThesedomesticspeciescouldnotoverwinteroutdoorsinHokkaido.Wilddrosophilidsshowaclear responsetoshortphotoperiodsandremainindiapauseforalongtime,atleastforamonthormoreat 15°C.Aweakresponsetoshortphotoperiods isalsoknowninsemi-domesticDrosophila(Sophophora) lutescens.InD. lutescens,thedevelopmentofovarieswasdelayedinshortdaylengthsat15°C,andthe 16-day-oldfemalesperfectlymaturedtheirovaries(Watabe,1985).Thespeciescannotsurvivethe winterofHokkaido.AshallowdiapauseofD. suzukiiand D. lutescenshasnoecologicalsignificancefor survivalintheseverewinterconditionsofHokkaido.OfthefivegeographicalstrainsofD. suzukii examinedatthistime,theSapporostrain(themostnorthernone)showedtheclearestresponse,andthe Shanghai strain (the most southern) was nearly neutral to short daylengths. In wild drosophilids distributedinsubtropicalregions,somespeciesorgeographicstrainsarenearlyneutraltophotoperiods (Kimura,1984).Intemperatewilddrosophilids,amorenorthernstrainundergeneticcontrolshoweda firmerorstrongerresponsetophotoperiods(cf.MinamiandKimura,1980;LummeandLakovaara,1982;
Kimura,1984).
Drosophila suzukiiiswidelydistributedfromthesubtropicaltothecooltemperateregions,andits weakincidenceofdiapausemayrelatetotheoriginalrangeofdistribution.Inawarmtemperateregion withamildwinter,aweakdiapausemaybeenoughtopermitthespeciestohibernatethere.Drosophila suzukiiisanoriginallywarmtemperatespecies,anditmighthavenotyetacquiredageneticmechanism Figure 6. PercentagesoftheDrosophilasuzukiifemaleswithundevelopedovariesrearedundershortandlong
daylengthsinfourdifferentgeographicstrains.
forafirmdiapause,whichcouldprovenecessaryforsurvivalduringthesevereandlongwinterofthe cooltemperateregion.
Figure7showsphotoperiodicresponsesofDrosophilasubpulchrella,asiblingspeciesofD. suzukiiin thesuzukiisubgroupoftheD. melanogasterspecies-group.ThespeciesisalsoaSino-Japaneseelement inthebiogeographyofDrosophila,andisdistributedfromsouthernChinatoHonshuinJapan(Okada, 1988)．AfirmincidenceofdiapausewasobservedinallthreestrainsofD. subpulchrellainLD:10:14at 15°Cand18°C.At18°C,mostofthe8-day-oldfemaleshadundevelopedovaries,andabouthalfofthe 16-day old females remained in diapause. Alower temperature (15°C) prolonged diapause. In the YakushimaandMatsuyamastrainsofD. subpulchrella,reproductivediapausewasmaintainedformore than2monthsunderLD10:14at15°C.Sucharemarkableordeepdiapausehasbeenfoundinmanywild drosophilidsdistributedinthewarmandcooltemperateregions.Thus,theweakincidenceofdiapause foundinD. suzukiiisaspecificeco-physiologicaltraitofthesuzukiisubgroup.
Cold-hardiness: Figure8AshowsthesurvivalpercentageofD. suzukii adults,whichwererearedat 15°Cfromitslarvalstageto8dayaftereclosion,keptat5°Cfor5days,andthenexposedtodifferent lowtemperatures(-1°Cto-6°C)for1day.Ingeneral,theadultsrearedunderashortdaylengthwere moreresistanttolowtemperaturesthanthoseunderalongdaylength.Stephenetal.(2015)studiedthe lowlethaltemperaturesofthesummermorph(rearedunderLD14:10at24°C)andthewintermorph (LD12:12at10°C)ofD.suzukii,andreportedthatmortalityoftheformerwaslowerthanthatofthe latter.Inthepresentexperiments,thesurvivalpercentageofadultsrearedundershortdaylengthswas about50%at-5°Cto-6°C,whereasitwasabout40%underlongdaylengths.Inthegraph,LT50(the temperatureatwhichhalfoftheadultsdead)wasbetweenat-4°Cto-5°C.Figure8Bshowsthe survivalpercentageofadultsthathadbeenrearedinLD16:8at15°Cor18°C,keptat5°C,andthen exposedto-4°C,-5°Cand-6°Cfor1day.Theadultsrearedat15°Cweremoretoleranttoextremelow temperaturesthanthoseat18°C.Theeffectoflow-temperature-rearingtocoldresistancewasreported inotherdrosophilidswithreproductivediapause,DrosophilaaurariaandD.lutescens(Kimura,1982a,b;
HiguchiandKimura,1985).Figure8Cshowsthesurvivalpercentageofadultfliesexposedto-2°Cand Figure 7. PhotoperiodicresponsesofthreegeographicstrainsofDrosophila subpulchrellarearedundershort
andlongdaylengthsat15and18 ℃ .Thefirmreproductivediapausewasinducedundershort daylengthsandmaintainedlongeratalowertemperature.A,Yakushima(⑪showninFigure1);B, Matsuyama(⑨);C,Okazaki(⑦).
-3°Cfor2days.At-2°C,thesurvivalpercentagesinLD10:14andLD16:8were73.0%and65.7%, respectively.At-3°C,theadultsrearedinLD10:14werealsomoretoleratetolowtemperaturesthan thoseinLD16:8,indicatinganeffectofashortphotoperiodsincold-hardiness.Figure8Dshowsthe survivalratein-2°Cfor1to3days.ThesurvivalrateofadultsrearedinLD16:8at15°Cwaslower thanthatinLD10:14. Mortality oftheadults inLD10:14at15°Cwasnearly constant for3days, suggestingthatmostoftheadultsremainedindiapause.
WeplacedtheglassvialscontainingD.suzukiiadultsunderthesnow(approximately30cmofdepth) inDecemberandcheckedthefly’sconditionafter2days.Theminimumtemperaturewasaround-7°Cin thosedays.Mostoftheadultssurvivedforoneday;92.6%(n=27)survivedintherearingconditionofLD 10:14and100%(n=30)inLD16:8.Noneofthefliessurvivedaftertwodays.Further,weplacedthevials withadultsinthesnowforonedayinmid-January(minimumtemperatureof-11.5°C),andnoneofthe adultssurvived.ThermaltoleranceofD. suzukii,especiallycold-hardiness,hasbeenstudiedintensively, mainlyfromtheviewpointofthemanagementofpestcontrolmanagement(Daltonetal.,2011;Stephens etal.,2015;Kaçaretal.,2016;Ryanetal.,2016;WallingfordandGregory,2016;Jakobsetal.,2017;Stockton etal.,2018).Thesestudiesclarifiedthat1)theadultstageismoretoleranttoextremetemperaturesthan itspre-imaginalstages,2)theadultsrearedinlowtemperaturesaremoreresistanttocoldstressthan thoseinhightemperatures,3)ashortdaylengthiseffectivetowithstandfreezingtemperatures,etc.
Figure 8. SurvivalpercentagesoftheDrosophilasuzukiiadultsexposedtovariouslowtemperaturesfor differentperiods.
Similarresultswereobtainedinthepresentstudy.
Theamountofadiposetissueaccumulatesandalmostoccupiesthewholeofabdomenindiapausing adultsofwilddrosophilids;thiswasnotobservedinD. suzukiifliescapturedinthefields(eveninlate autumn).Mostspeciesofwilddrosophilidsoverwinterinhibernationsitessuchastreeholes,cliff- sheltersalongstreams,andgrass-stands.Drosophila suzukiimustavoidextremecoldtemperatureto survivethroughtheharshclimate,butstillvisitstrapsuntilmid-Novemberanddoesnotentersuitable hibernationsites.Drosophila suzukii doesnotinvadehouses,unlikedomesticspeciesof D. melanogaster and D. busckii. Thus, the Sapporo population of D. suzukii is exterminated by harmful freezing temperaturesduringthewinterandisre-establishedeveryyearfromfoundersthatemigratefrom southerndistrictsofJapaninthenextspring.
IntheKantoandShikokudistrictswithfewsnow(⑥,⑨,and⑩inFig.1),D.suzukiiwascollected duringthewintermonths(DecembertoFebruary)bybananatrapsandnet-sweepings(Toda,1979;
Takamori,1997).There,thespeciesvisitstreesapsforfeedingsinthewinter.Thus,itisreasonableto assume that D. suzukii does not remain in reproductive diapause in these regions. Howeer, in Mt.
Dakesan(⑫)oftheShimanePrefecturenearShikoku,D. suzukiiwasabsentorinextremelylow numbersduringAprilandMay(Wakahama,1962).Themountainhasmuchsnowcausedbythestrong windfromthenorthwestduringthewinter,andsoitisthoughtthatD. suzukiicannothibernatethere.
Drosophila suzukii cansurvive through the winter in the plains of Kyushu, but not in its central highlands.AprobableborderlineforoutdoorhibernationofD. suzukiiispresentedinFigure1basedon the present experiments on cold-hardiness and the winter field collections mentioned above. The borderlineliesbetweennorthernKantoandsouthernTohoku.Thesedistrictsarethetransitionzoneson thefloraofJapan,fromwarmtemperateevergreenforests,tocooltemperatedeciduousbroad-leaved forests.ThearearangewhereD. suzukiispendsthewinteroutdoorsisalmostoverlappedtothatof evergreenforestsfromNanseiIslandstoKanto(Kamijo,2009).AlthoughashallowdiapauseofD. suzukii maynothaveanyadaptivesignificanceonthesafeoverwinteringinsevereclimatesofHokkaido,itmay beeco-physiologicallymeaningfularoundthenorthernbordersofdistribution,northernKantoand southernTohoku,becauseD.suzukiiadultsthatexperiencedshortdaylengthsaretoleranttosudden changesintemperatureandcanmoreorlessevadevainproductionsinunsuitabledays.Naturalselection mayreinforceafirmerresponseforD. suzukii undershortphotoperiodsintheseregions.Inrecentyears, someofthemeteorologicalstationsrecordatendencyofaminimumtemperatureriseduringthewinter, probablyowingtoinfluencesofglobalwarming,andaccordingly,theborderlineforoverwinteringofD.
suzukii mayshifttothenorth.
TheoriginoftheHokkaidopopulationofD. suzukii isstillopenforquestion;forexample,fromwhich districtsdoesD. suzukiiimmigratetoHokkaido.WestillhavenoknowledgeonhowD.suzukiiarrivesin Hokkaido, although there are several possibilities; with commercial fruits transported from warm districts,withtheaidofthesouthernwind,orothers.WearelookingforthefounderoftheHokkaido populationofD. suzukii.
４．ACKNOWLEDGEMENTS
WearegratefultoDr.Fuyama,Y.(theEmeritusProfessorofTokyoMetropolitanUniversity),who kindly provided us with geographic strains of D. suzukii and D. subpulchrella for the laboratory experiments.WealsothankDr.Ryushima,H.(HokkaidoUniversityofEducation:HUE),Mr.Nakamura, K.(Sapporo)andMiss.Tamura,M.(HUE)fortheirfacilitiesinfieldstudies.TheStatisticsBureau, MinistryofInternalAffairsandCommunicationsofJapan,provideduswiththemeteorologicaldataused foranalysesontheannualfluctuationofD. suzukiipopulations.
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（神田 侑奈 札幌校大学院生）
（並川 寛司 札幌校教授）
（渡部 英昭 北海道大学総合博物館研究員）
