Table Of Content©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
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