©EntomologicaFennica.5December2007 Transmission of the pine wood nematode Bursaphelenchus xylophilus through oviposition activity of Monochamus galloprovincialis (Coleoptera: Cerambycidae) PedroM.Naves*,SusanaCamacho,EdmundodeSousa&JoséA.Quartau Naves,P.M.,Camacho,S.,Sousa,E.&Quartau,J.A.2007:Transmissionofthe pinewoodnematodeBursaphelenchusxylophilusthroughovipositionactivityof Monochamusgalloprovincialis(Coleoptera:Cerambycidae).—Entomol.Fen- nica18:193–198. Transmission of Bursaphelenchus xylophilus (PWN) by the oviposition of MonochamusgalloprovincialiswasstudiedinPortugal.Femaleinsectslaideggs on 77% of pine bolts on the laboratory, of which 37% became infected with PWN, with a mean of 290 nematodes. Inside the PWN affected zone 50 P.pinastertrap-treeswerecreatedbetweenMayandSeptember2001and2002, ofwhich29werecolonisedbyM.galloprovincialisandonly fourbecamein- fectedbythenematode.Thelowtransmissionefficiencydetectedbothonlabora- tory and field suggests that nematode transmission through the vector’s ovipositionactivitymaybeasecondarycomponentofthepinewiltdiseaseepi- demiologyinPortugal. P. M. Naves, S. Camacho & E. de Sousa, Dep. Protecção Florestal, Estação FlorestalNacional–INIAP.QuintadoMarquês2780–159Oeiras,Portugal; *correspondingauthor’se-mail:[email protected] J.A.Quartau,FCUL,CentrodeBiologiaAmbientaleDep.BiologiaAnimal, CampoGrande,EdifícioC2,1749-016Lisboa,Portugal Received17July2006,accepted20March2007 1.Introduction Transmission through the feedings activity occurs when the recently emerged adult insects Transmissionofthepinewoodnematode(PWN) make their maturation feeding in the bark of Bursaphelenchus xylophilus (Steiner & Buhrer) healthy conifer trees (Mamiya & Enda 1972, Nicklefromitsinsectvectortoatreehostisanes- Wingfield&Blanchette1983,Luzzietal.1984, sential component of the epidemiology of the Linit 1990). The B. xylophilus JIVlarvae aban- pinewiltdisease.Worldwide,themostimportant dontheinsect’sbodyandenterthehostthrough PWNinsectvectorsarecerambycidbeetlesofthe thewoundsmadethebeetlewhenfeedingonthe Monochamus genus (Linit 1988, Kishi 1995), conifer’sbark.Ifthehostissusceptibletothepine which carry the fourth-stage dispersal juvenile woodnematodetheinoculatedindividualsbegin nematodesintonewtreehosts.Nematodetrans- tofeedonthexylemtissuesandreproduce,even- mission occurs mainly in two distinct ways: tually interrupting the water and resin flow and through the adult’s feeding activity in healthy killingthetree(Linit1988).Incontrastwiththe treesorthroughthefemales’ovipositionwounds previouspathway,transmissionthroughtheovi- (Linit1990). positionwoundsoccurswhenthefemaleMono- 194 Navesetal. (cid:127) ENTOMOL.FENNICAVol.18 chamus choose dead or weakened trees (or re- freshmaritimepinebranchesforfeeding(15cm cently-cutlogs)tolaytheireggs(Wingfield1983, long,0.5–1cmdiameter)andsomepinebolts(10 Wingfield&Blanchette1983,Luzzietal.1984, cmlong,4–5cmdiameter)forlayingeggs.When Linit1989,1990,Edwards&Linit1992).When they attained 30 days of life [age which corre- the female insects chew the bark to insert their sponds roughly to the oviposition peak of this eggs,thenematodesleavetheirbodiesandenter species(Navesetal.2006)],themaleswerere- the decayed or dead new host through the movedfromtheboxesandeachfemalewasgiven ovipositionholes. asingleP.pinasterbolt(10cmlong,4–5cmdi- InPortugal,wherethePWNwasdetectedin ameter) for laying eggs, along with two addi- 1999, the endemic pine sawyer Monochamus tional0.5–1cmwidepinebranchestofeed.The galloprovincialis(Olivier)isitssolevector(Sou- feeding and oviposition bolts were obtained six saetal. 2001), and therecently detected nema- daysearlierfrom10-yearoldhealthyP.pinaster tode-vectorassociationhasbeensubjectedtosev- treescutonOeiras,outsidetheB.xylophilusaf- eral studies, namely the characterisation of the fectedzone. transmission by the insect’s feeding activity Eachboltremainedwiththefemalefor24h, (Navesetal.2007).Thepurposeofthisstudywas afterwhichwasreplacedbyasimilarone,total- to characterise a second possible transmission lingfourreplicas(bolts)perbeetle.Afterthere- pathway,thatis,bytheovipositionactivityofthe movalofthelastbolt,thefemalesweremeasured femaleM.galloprovincialis,determiningitsoc- (lengthoftherightelytra),weightedandmacer- currence and frequency under laboratory and atedonaPetridishwithwater,countingthenum- fieldconditionsanddiscussingtheimportanceof ber of nematodes present in the water after 12 thistransmissionpathway fortheepidemiologi- hours. calcycleofthepinewiltdiseaseinPortugal. Oviposition wounds on bolts were counted and debarked, to determinate if they contained eggs. If present, feeding scars on the bark were 2.Materialsandmethods countedandremovedwithaverticalelectricsaw, alongwithapiece(2–3cm)ofadjacentwood,in 2.1.Nematodetransmission order to prevent the contamination of the wood underlaboratoryconditions withnematodeseventuallyintroducedduringthe female’s feeding activity. The pine bolts were Three adult maritime pine trees (Pinus pinaster thenstoredinclosedplasticbagsat25ºC±1ºC Aiton) infected with B. xylophilus and M. and75%RHfortwoweeks,toallowtherepro- galloprovincialiswerecutinJanuary2004near ductionofthenematodesintroducedonthewood Comporta (38º 22’N; 8º 46’W), inside the pine and increase the probability of extracting them, wood nematode affected zone in Portugal. The which was made using the modified Baermann trunksweredividedinto70cmboltsandmain- technique (Southey 1986). Direct counts were tainedunderambienttemperatureonnettedwood made for samples lower that 500 nematodes, boxes until emergence of adult M. gallo- while for higher nematode values a counting provincialis in the following spring. Recently- lamellaewith1mlofthesamplewasused,repli- emerged beetles were immediately checked for catingthecountfivetimesandextrapolatingthe thepresenceofB.xylophilususingthemethodol- meanofthefivereplicastotheinitialsamplevol- ogydescribedbyZhangetal.(1995). ume. Fifteen female beetles found to be infected Spearman correlations were performed to with PWN were placed isolated on transparent analyse the relation between insect dimensions acrylic boxes (volume of 3100 cm3), to which (sizeandweight)andnematodeload,andalsobe- were added two adult males of similar size and tween PWN abundance and number of ovi- age.Theboxes,containingseveralsmallholesfor positionwounds(withandwithouteggs),feeding air renewal, were kept in a room with a stable woundsandinsectnematodeload.Statisticalpro- temperature of 23–25ºC, 15:9 LD and 60–75% cedures were carried out using the software humidity.Everytwodaystheinsectsweregiven Statistica6(StatSoftInc.2003). ENTOMOL.FENNICAVol.18 (cid:127) PWNtransmissionbypinesawyeroviposition 195 2.2.Nematodetransmission Table1.MeannumberofM.galloprovincialis’feeding underfieldconditions andovipositionwoundsperboltandmeannumberof B.xylophilusrecoveredfromthebolts.n=60bolts. Meanvalues±S.E. Maritime pine trap-trees were set inside Tróia (38º28’N;8º52’W)andComp.dasLezírias(38º Feeding Ovipositionwounds No. 47’N; 8º 49’W) inside the PWN affected zone, wounds PWN Portugal. Each trap consisted of an apparently Withouteggs Witheggs healthyadultP.pinastertree(mean18cmchest height diameter) debarked at waist height, re- 0.78±0.15 0.70±0.19 1.37±0.22 290±70 movingalongsidepartofthecambiumandxylem woodtocompletelystoptheresinflow.Theob- jective was to slowly debilitate and eventually killthetree,thusturningitintoahighlyattractive NorelationwasfoundbetweenB.xylophilus hostforovipositionbythelocalMonochamuspo- woodabundanceandthenumberofinsectfeed- pulation. An initial sample of wood from each ingwounds(r=0.208,p=0.11)oroviposition tree was taken at breast height and analysed on wounds without eggs (r = –0.118, p = 0.37). thelaboratoryusingthemodifiedBaermanntech- Therewasamodest(butsignificant)correlation nique to ensure that the pines were nematode- betweenthenumberofovipositionwoundswith free. The trap-trees were created between May eggs and nematode abundance (r = 0.575, and September of 2001 (two trap-trees for each p<0.001). monthandlocation)and2002(threetrap-treesfor AllthedissectedMonochamusfemaleswere eachmonthandlocation),inatotalof50treesfor foundtocarryB.xylophilusontheirbodies,with both years. In November of each year the trees ameanvalueof1684±516(mean±S.E.)nema- werecut,dividedinonemeterlogsandtakento todesperinsect.Themeanlengthofthe15female the Lab, where they were analysed (trunk and beetles was 15.2 ± 0.4 mm, with a weight of branches) for the presence of M. galloprovin- 0.3868±0.023g(meanvalues±S.E.),anditwas cialis oviposition and feeding scars. Two wood found that the smaller beetles carried a larger discs were collected at breast height and mid- number of nematodes than the larger ones, as crownfromeachtreeandanalysedforthepres- modestnegativecorrelationswerefoundbetween ence of B. xylophilus using the modified Baer- thenematodeloadandbeetlesize(r=–0.432,p= manntechnique. 0.11)andweight(r=–0.566,p=0.028),being the second correlation significant. No relation was found between the insect’s nematode load 3.Results and PWN abundance on wood (r = 0.079, p = 0.55). 3.1.Nematodetransmission underlaboratoryconditions 3.2.Nematodetransmission Of the 60 bolts analysed, 46 (77% of the total) underfieldconditions contained a total of 124 M. galloprovincialis ovipositionwounds,ofwhich66%hadeggs.De- Allthewoodsamplescollectedinitiallyfromthe spite the presence of small pine branches ade- pinestestednegative,andthustheselectedtrees quateformaturationfeeding,38%oftheboltsin- wereconsideredtobenematode-freeatthedate tendedforovipositionalsohadmaturationfeed- oftrap-treecreation.Overthetwoyears,29ofthe ingscars(Table1). 50 pines (58%) were colonised by M. gallo- Only 37% of the bolts contained nematodes provincialis, with a total of 439 oviposition and usually with low values, as the mean was woundsdetected(Table2). lowerthat300individualsperbolt(Table1).B. Despite the abundant presence of Mono- xylophiluswastheonlynematodepresentonthe chamusreproductiveactivity onthemajority of wood. thepines,onlyfourofthetreeswithsawyerpres- 196 Navesetal. (cid:127) ENTOMOL.FENNICAVol.18 Table2.Numberoftrap-treescolonisedbyM.galloprovincialis(M.g.)onthetwolocations(LezíriasandTróia), totalnumberofM.g.ovipositionwoundsonthetrees,numberoftreesinfectedwithB.xylophilusandmean numberofPWNper100gofwood.Thereweretwoandthreetrap-treesin2001and2002,respectively,ineach monthandlocation. Lezírias Tróia Date No.trees No.M.g. No.trees Mean No.trees No.M.g. No.trees Mean (month/ withM.g. wounds withPWN PWN/ withM.g. wounds withPWN PWN/ year) 100g 100g May/01 2 41 0 – 2 120 0 – May/02 1 27 0 – 1 5 1 116 June/01 2 54 1 833 1 8 1 477 June/02 2 6 0 – 2 11 0 – July/01 1 31 0 – 1 9 0 – July/02 3 19 0 – 2 2 0 – Aug/01 2 10 0 – 2 40 0 – Aug/02 0 – 0 – 0 – 0 – Sept/01 2 22 0 – 0 – 0 – Sept/02 2 32 0 – 1 2 1 56 Total 17 242 1 – 12 197 3 – encewerefoundcontainingB.xylophilus,witha tively low, and reflect a low abundance of meanof371nematodesper100gofwood.The Monochamusfemalesreproducingonthetwoar- pinewoodnematodewastheonlyspeciesofthe easselectedand/oranabundanceofoviposition- Bursaphelenchusgenuspresentonthewood,al- suitablepinehosts,whichdispersethereproduc- thoughotherAphelenchoididaewerealsofound tiveactivityofthefieldinsects.Thismightalso onsometrees. explaintheverylownumberofeggslaidonthe September trap-trees, which probably reflect a decreaseintheinsectpopulationduringlatesum- 4.Discussion merinparallelwithasignificantaugmentonthe numberofnaturallyoccurringdecayinganddead Thelaboratoryexperimentconfirmedthattrans- pinetreesadequateforoviposition. missionofB.xylophilusthroughtheoviposition Itisnotpossibletodeterminatethenumberof activityoffemaleM.galloprovincialiscanoccur. insects that visited the trap-trees to lay eggs, or Someoftheovipositionboltsalsocontainedad- knowtheirnematodeinfectionrate,butanalysis ditionalfeedingwoundsandovipositionwounds offieldcollectedbeetlescapturedonbothloca- without eggs, but bolt nematode abundance ap- tions show that usually about 70% of the field- pearstoberelatedtotheexistenceofoviposition captured insects are infected with B. xylophilus wounds with eggs. The transmission efficiency (Sousa,unpubl.).Asso,itisreasonabletoassume wasratherlow,though,asonly37%ofthelabora- thatasignificantpartoftheinsectsthatvisitedthe toryboltsand14%ofthefieldtrap-treesbecame pinetreeswereinfectedbyB.xylophilus,despite PWN infected, and usually with low nematode thefactthatonlyfourofthetreesbecamecontam- numbers(lessthan100nematodesperboltforthe inated by PWN. The possibility of additional Labexperiment,despitetheboltshadbeenkept nematode transmission occurring through the onfavourableconditionsforB.xylophilusrepro- feedingactivityofthebeetlescannotbeexcluded, ductionduring15consecutivedays).Concerning butitwasimpossibletoevaluateasthetreeshad thefieldexperiment,thepercentageoftrap-pines multiplebark-feedingscarsmadebydifferentin- with M. galloprovincialis oviposition wounds sect species (cerambycidae, curculionidae and (58%) and the total number of oviposition scolytidae), which cannot be discriminated by wounds detected (439) can be considered rela- species. ENTOMOL.FENNICAVol.18 (cid:127) PWNtransmissionbypinesawyeroviposition 197 M. galloprovincialis has been found to suc- through feeding activity (Naves et al. 2007), cessfullytransmitB.xylophilusbythefeedingac- transmissionofB.xylophilusthroughoviposition tivity on pine branches under laboratory condi- woundsisaninfrequentandsecondaryinocula- tions(Navesetal.2007).Thisisthefirstrecordof tion pathway and probably constitutes a minor PWNtransmissionviaM.galloprovincialisovi- componentofthepinewiltdiseaseepidemiology. position under laboratory conditions, although thistransmissionpathwayhasbeenreportedfor Acknowledgements. We thank Mr. Rui Andrade for his several North American Monochamus species helpinobtainingthetreesandananonymousreviewerfor itscomments.TheworkwaspartiallyfundedbytheFoun- (Wingfield1983,Wingfield&Blanchette1983, dationforScienceandTechnology(PortugueseMinistry Luzzietal.1984,Edwards&Linit1992).Trans- ofScienceandTechnology)throughtheprojectParleD, missionviaovipositionisprobablyanevolution- and also the European Phrame project QLK5-CT-2002- aryadaptationforavirulentnematodestopersist 00672. inforestecosystems,asthenematodesareinocu- latedbytheirvectorsinalreadydeadorweakened References hosts,andthusdonotneedtokillthetreeinwhich they are inoculated (Wingfield & Blanchette 1983). As for B. xylophilus, this transmission Bergdahl, D. 1988: Impact of pinewood nematode on NorthAmerica:presentandfuture.—J.Nematol.20: pathway probably evolved as an adaptation to 260–265. NorthAmericanpineecosystems,wherethepine Edwards, O. & Linit, M. 1992: Transmission of Bursa- woodnematodeusuallydoesnotcausepinewilt phelenchusxylophilusthroughovipositionwoundsof on indigenous pines, being generally associated Monochamus carolinensis (Coleoptera: Cerambyci- withtreessufferingfromtheactivityofotherbi- dae).—J.Nematol.24:133–139. Kishi,Y.1995:ThepinewoodnematodeandtheJapanese otic or abiotic decline agents (Wingfield et al. pinesawyer. —ThomasCompanyLimited, Tokyo, 1982, Wingfield 1983, Rutherford & Webster Japan.302pp. 1987,Bergdahl1988). Linit,M.1988:Nematode-vectorrelationshipsinthepine Similarly to other Monochamus species, fe- wiltdiseasesystem.—J.Nematol.20:227–235. male M. galloprovincialis only lay eggs on al- Linit, M. 1990: Transmission of pinewood nematode throughfeedingwoundsofMonochamuscarolinensis readydeadordyingconifers,ofwhichthereisan (Coleoptera:Cerambycidae).—J.Nematol.22:231– abundant supply on the Portuguese PWN af- 236. fectedzoneduringthelatesummermonthsdueto Luzzi,M.,Wilkinson,R.&Tarjan,A.1984:Transmission previous B. xylophilus infection. Other locally ofthepinewoodnematode,Bursaphelenchusxylophi- important pine mortality agents include cyclic lustoslashpinetreesandlogboltsbyacerambycid beetle,Monochamustitillator,inFlorida.—J.Nema- heavydefoliationbythepineprocessionarymoth tol.16:37–40. [Thaumetopoea pityocampa (Den. & Schiff.)], Mamiya,Y.1984:Thepinewoodnematode.—In:Nickle, forestfires,barkbeetles(curculionidaeandsco- W. R. (ed.), Plant and insect nematodes: 589–626. lytidae) and drought, but, with the exception of MarcelDekkerPress,NewYork. forestfires,theothermortalityagentsexerttheir Mamiya, Y. & Enda, N. 1972: Transmission of Bursa- phelenchuslignicolus(Nematoda:Aphelenchoididae) action mainly on young, dominated and low- byMonochamusalternatus(Coleoptera:Cerambyci- sized pines, which are usually not colonised by dae).—Nematologica18:159–162. M. galloprovincialis nor B. xylophilus (Sousa, Naves, P., Sousa, E. & Quartau, J. 2006: Reproductive unpublishedresults).Asso,alargeproportionof traitsofMonochamusgalloprovincialis(Coleoptera; theMonochamusfemaleslaytheireggsonpines Cerambycidae)underlaboratoryconditions.—Bull. Entomol.Res.96:289–294. already contaminated by PWN. 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