Molecular identification of Trichogramma species from South and South-East Asia and natural Wolbachia infection

2019·0.35 MB·

by  Yu-Di Liu

©EntomologicaFennica.16June2017
Molecular identification of Trichogramma species from
South and South-East Asia and natural Wolbachia infection
Yu-DiLiu,Mao-LinHou&KaiSong
Liu, Y.-D., Hou, M.-L. & Song, K. 2017: Molecular identification of Tricho-
grammaspeciesfromSouthandSouth-EastAsiaandnaturalWolbachiainfec-
tion.—Entomol.Fennica28:57–66.
Trichogrammawaspswerecollectedfromtheparasitizedeggsoflepidopteran
pests from 21 sampling sites in East Asia and South-East Asia. Six Tricho-
grammaspecieswereidentifiedbasedonthemolecularidentificationmethodus-
ingtheinternaltranscribedspacer2(ITS2)regionoftherDNAofTrichogramma
chilonis, T. evanescens, T. ostriniae, T. embryophagum, T. dendrolimi and T.
japonicum.Theresultsofmolecularidentificationwereconfirmedbymorpho-
logical identification. Additionally, natural populations were screened for the
prevalenceofWolbachia.Fiveoutof21populationswereinfectedbythesame
Wolbachiastrain,whichwasidentifiedbyusingWolbachiawspgeneandmulti–
locus sequencing approach. The phylogenetic analysis of Wolbachia wsp se-
quencesrevealedthattheWolbachiastrainwasclassifiedinthestrainwEvaAin
thegroupofEvAofthesupergroupA.
Y.-D.Liu*&M.-L.Hou,StateKeyLaboratoryforBiologyofPlantDiseasesand
InsectPests,InstituteofPlantProtection,ChineseAcademyofAgriculturalSci-
ences,No.2,WestYuanMingYuanRoad,Beijing100193,China;*Correspond-
ingauthor’se-mail:[email protected]
K.Song,DrylandFarmingInstitute,HebeiAcademyofAgriculturalandFor-
estrySciences,Hengshui,China
Received15December2015,accepted11November2016
1.Introduction izedskills(Pintoetal.1989,Pinto&Stouthamer
1994, Poorjavad et al. 2012). In order to be of
Thereareabout650speciesinthefamilyTricho- economic importance in biological control pro-
grammatidae(Grissel&Schauff1990),andthey jects, it is essential to be able to identify the
arethemostwidelyusedparasitoidsinbiological Trichogrammatid species quickly, and the me-
control programs (Kumar et al. 2009). For suc- thodshavetobesimpleandwidelyapplicable.
cessfulbiologicalcontrolpurpose,theidentifica- Molecular approaches based on DNA se-
tion of Trichogramma species is the important quences of the internal transcribed spacer 2
first step (Hassan 1994). Unfortunately, species (ITS2)havehelpedtosolvetheabovedifficulty
identificationinthisgroupisdifficultduetotheir (Stouthamer et al. 1999). Several studies have
small size, the large number of species, and the usedtheITSregiontoidentifytheTrichogramma
lackofclearmorphologicalcharacteristics.Iden- speciesoccurringindifferentregions(Silvaetal.
tificationistimeconsumingandrequiresspecial- 1999, Kumar et al. 2009, Sumer et al. 2009,
58 Liuetal. (cid:127) ENTOMOL.FENNICAVol.28
Poorjavadetal.2012,Nasiretal.2013,Pinoetal. 2.Materialsandmethods
2013).TheITS2regionhasbeenusedtodistin-
guish Trichogramma species collected from to- 2.1.Trichogrammacollection
matofieldsinPortugalbysequencingandrestric-
tionanalysis(Silvaetal.1999).Pinoetal.(2013) Parasitized Trichogramma eggs were collected
rapidlyidentifiedfiveTrichogrammaspeciesoc- from21paddyfieldsinChinaandKoreain2011
curringintheCanaryIslandsbyusingmultiplex and2012(Table1),andkeptindividuallyinglass
PCRmethodbasedonamplificationofITS2re- tubesuntiladultemergence.Insomefields,fresh
gion.Poorjavadetal.(2012)usedPCRamplifi- sentineleggsofCorcyracephalonica(Stainton)
cation of ITS2 region to identify seven Iranian (Lepidoptera: Pyralidae) were used to trap
Trichogramma species, which were identical in Trichogramma.Therefore,theoriginatinghostof
externalmorphology.Nasiretal.(2013)usedthe Trichogrammaforsomesitesisthesentinelhost
ITS2 region of rDNAto distinguish six Tricho- C.cephalonica.Aftercollection,theadultswere
grammaspeciescollectedfromdifferentecologi- reared on eggs of the grain moth, Sitotroga
calzonesofPakistan. cerealella Olivier (Lepidoptera: Gelenchiidae),
InEastAsiaandSouth-EastAsia,rice(Oryza in climatechambers at 25 ± 1 °C, 70 ± 5% RH
sativa L.) is a staple food source for more than with the photoperiod of 14:10 (L:D) h. Popula-
half of the world’s population (Gross & Zhao tions are defined as the progeny from one egg
2014).Toreducetheapplicationofpesticidesin batch,whicharecollectedfromthesamespecies
controlofthericepests,integratedpestmanage- atthesamelocationonthesameday.
ment (IPM) based on biological control by
Trichogrammareleaseswaslaunched(Koetal.
2.2.DNAisolation
2014). Therefore, in this study, comprehensive
fieldsurveyswereconductedintheseregionsto DNA was extracted using chelating agent
collect Trichogramma species. Finally, Tricho- Chelex–100(5%)(Biorad)methodaccordingto
grammawerecollectedfrom21samplingsitesin Stouthameretal.(1999).Onetothreewaspsfrom
East Asia and South-East Asia. The molecular thesingleparasitizedeggweregroundin100µl
methodbasedonITS2regionwasusedtoidentify 5%Chelex–100(Biorad)and3µlproteinaseK
theseTrichogrammaspecies. (20 mg/ml) and incubated for 2 h at56 °C, fol-
Ontheotherhand,Wolbachia,asasymbiotic lowedby10minat95°C.Thesupernatantwas
bacterium, plays important roles in evolution, storedat–20°Cforsubsequentmolecularanaly-
ecology,andreproductionoftheirhosts(Werren sis.
1997). They are extremely common, with 20–
76%ofinsectspeciesbeinginfected(Harrisetal.
2.3.PCRamplification
2003).Morethan20Trichogrammaspecieshave
beenpartlyorcompletelyinfectedbyWolbachia TheITS2regionwasamplifiedusingthefollow-
(Poorjavad et al. 2012). Because Wolbachia in- ing primers: forward, 5 –TGTGAACTGCAG-
fectioncanaffectthewasp’sfitness,itisneces- GACACATG–3,locatedinthe5.8SrDNA;and
sarytoinvestigatetheinfectionstatusofapopu- reverse, 5 –GTCTTGCCTGCTCTGAG–3, lo-
lation, which may give important aids for catedinthe28SrDNAclosertothe3endofthe
Trichogramma application in biological control ITS2(Stouthameretal.1999).Touchdownther-
programs (Stouthamer & Kazmer 1994, Horjus malcyclingprogramsencompassinga5°Cspan
& Stouthamer 1995, Poorjavad et al. 2012). ofannealingtemperaturesat55–50°Cwereper-
Therefore, in this study, the collected Tricho- formed for the amplification using a S1000™
gramma wasps were screened for the infection ThermalCycler(Bio–Rad).Afteraninitialdena-
with Wolbachia, and the respective Wolbachia turation at 94 °C for 4 min, cycling parameters
strainwasidentifiedbysequencesofWolbachia were10cyclesof95°Cfor20s,highestanneal-
surface protein (wsp), Cytochrome c oxidase, ingtemperature(decreased0.5°Cpercycle)for
subunit I (coxA) and Fructose-bisphosphate 30s,and72°Cfor30s;and30cyclesof95°Cfor
aldolase(fbpA). 20s,lowestannealingtemperaturefor30s,and
ENTOMOL.FENNICAVol.28 (cid:127) MolecularidentificationofTrichogramma 59
Table1.OriginsofTrichogrammaspeciesandstrainswithoriginatinghosts,no.ofbasepairsinITS2(internal
transcribedspacer2regionofrDNA)sequencesandGenBankaccessionnumbers.
Sampleno. Species Population Host ITS2and
andCode Acc.no.
1.Tc-CJ T.chilonis Cuijia,Xing’an,China Corcyracephalonica 426
34°55’46”N,109°37’51”E KR148947
2.Tc-WLP T.chilonis Wulipai,Xing’an,China C.cephalonica 426
24°24’0”N,120°37’58”E KR148947
3.Tc-Lo T.chilonis Vientiane,Laos Chilosuppressalis 426
17°58’0”N,102°36’0”E KR148948
4.Tc-NB T.chilonis Ningbo,China C.suppressalis 426
29°52’48”N,121°33’0”E KR148948
5.Tc-MM T.chilonis Hmawbi,Myanmar Scirpophagaincertulas 426
17°5’0”N,95°57’0”E KR148948
6.Tc-TW T.chilonis Taiwan,China C.suppressalis 426
25°3’0”N,121°31’0”E KR148948
7.Tc-HS T.chilonis Hengshui,China C.suppressalis 426
37°32’14”N,115°28’59”E KR148949
8.Te-HS T.evanescens Hengshui,China Ostriniafurnacalis 546
37°32’14”N,115°28’59”E KR148950
9.Te-BJ T.evanescens Beijing,China C.cephalonica 546
39°54’27”N,116°23’17”E KR148950
10.Te-CQ T.evanescens Chongqing,China C.cephalonica 546
29°10’47”N,106°9’36”E KR148950
11.Te-K1 T.evanescens Korea O.furnacalis 546
37°28’N,126°37’E KR148950
12.Te-K2 T.evanescens Korea O.furnacalis 546
37°28’N,126°37’E KR148950
13.Te-Tconf T.evanescens Guangdong,China O.furnacalis 546
23°4’48”N,113°8’24”E KR148951
14.Te-GD T.evanescens Guangdong,China O.furnacalis 544
23°4’48”N,113°8’24”E KR148952
15.To-HBZ T.ostriniae Hebianzai,Dehong,China C.cephalonica 566
24°16’57”N,104°25’21”E KR148945
16.To-MZ T.ostriniae Mangzai,Dehong,China C.cephalonica 566
24°0’16”N,101°6’26”E KR148945
17.To-HS T.ostriniae Hengsui,China C.cephalonica 566
37°32’14”N,115°28’59”E KR148946
18.Tem-Tcac T.embryophagum Husa,Dehong,China C.cephalonica 587
24°27’48”N,97°53’24”E KR148953
19.Td-NJ T.dendrolimi Nanjing,China Cnaphalocrocismedinalis 520
32°3’0”N,118°46’60”E KR148954
20.TJ-GD T.japonicum Guangdong,China C.suppressalis 432
23°4’48”N,113°8’24”E KR148955
21.Tj-HS T.japonicum Husa,Dehong,China S.incertulas 432
24°27’48”N,97°53’24”E KR148955
72°Cfor30s.Thiswasfollowedbyafinalexten- fromTrichogrammasampleswasthatdescribed
sionstepat72°Cfor7min.Eachreactionwasrun by Braig et al. (1998). We also used two con-
inavolumeof30µL,containing3µL(10×)Taq servedWolbachiagenes,fbpAandcoxA,tochar-
assay buffer, 300 µM dNTP, 0.4 µM of each acterize Wolbachia strain for Trichogramma
primer, 1 U of Taq DNA polymerase (TaKaRa samples,whichwereamplifiedbasedontheme-
Biotechnology, Dalian, China), and 50–100 ng thods of Baldo et al. (2006). The PCR primers
genomicDNA. were designed also according to Baldo et al.
Theprimerusedtoamplifythewspfragment (2006). The PCR reaction conditions and the
60 Liuetal. (cid:127) ENTOMOL.FENNICAVol.28
Table2.InformationofwspgeneforconstructingthephylogenictreeofWolbachiastrainsinFig1.
Group Wolbachiahostspecies Wolbachia GenBank
strain Acc.no.
SupergroupA
Ha Drosophilasechella wHa AF020073
Ha Cadracautella wCauA AF020075
Aus Glossinaausteni wAus AF020077
Kue Trichogrammaourarachae wBou AF071913
Kue Trichogrammaevanescens wEvaB AY390280
Kue Trichogrammakaykai wKayA AF071912
Mel Drosophilasimulans wCof AF020067
Mel Amitusfuscipennis wFus AF071909
Mel Drosophilamelanogaster wMel AF020063
Dro Trichogrammadrosophilae wDro AF071910
Eva Trichogrammaevanescens wEvaA AY390279
Pap Phlebotomuspapatasi wPap AF020082
Uni Muscidifuraxuniraptor wUni AF020071
Mors Nasoniavitripennis wVitA AF020081
SupergroupB
Ori Leptopilinaaustralis wAus AF071920
Ori Cadracautella wCauB AF020076
Ori Spalangiafuscipes wFu AF071921
Con Trichogrammabedeguaris wBed AF071915
Dei Trichogrammadeion wDei AF020084
Vul Armadillidiumvulgare wVul AF071917
Pip Trichogrammachilonis wChi AY311486
Pip Drosophilasimulans wMa AF020069
Pip Culexpipiens wPip AF020061
Spodopteraexigua wExiB EU332344
Pip Ostriniafurnacalis wFurB EU294312
Kay Trichogrammakaykai wKayB AF071924
Kay Trichogrammanubilale wNub AF071926
SupergroupD
Brugiamalayi JX506736
SupergroupF
Cimexlectularius DQ842459
thermalcyclingprotocolwereidenticaltothose aligned with Clustal X1.83 (www.clustal.org).
describedabove. Some reference sequences of Wolbachia wsp,
After purified with PCR Cleanup Kit coxA,andfbpAsequencesofotherspecieswere
(Axygen,USA),thePCRproductsweredirectly downloadedfromGenBankforthephylogenetic
sequenced on an Automated DNA Sequencer analysisofWolbachia(Tables2and3).Inorder
(ABIPRISMTM 3730XL, APPLIEDBIOSYS- toverifytheconsistencyofthetree,phylogenetic
TEMS, INC. Foster City, CA). All sequences treeswereconstructedusingtwomethods,neigh-
werealignedusingCLUSTAL_X(Thompsonet borjoining(NJ)andmaximumparsimony(MP)
al.1997)andrecheckedbyeyetoverifyforaccu- (Mega 4.0 software, MEGA, www.megasoft-
racy. ware.net). Bootstrap analysis was done with
1,000replications,andbootstrapvalueswerecal-
culatedusinga50%majorityrule.Tworeference
2.4.Phylogeneticanalysis
sequences belonging to the Wolbachia D and F
Thewsp,coxA,andfbpAsequencesofWolbachia supergroups were used as the outgroups in the
from the different Trichogramma populations phylogenetic trees of wsp, coxA, and fbpA;
were first blasted in NCBI, then analyzed and Brugia malayi (Brug, 1927) (wsp JX506736,
ENTOMOL.FENNICAVol.28 (cid:127) MolecularidentificationofTrichogramma 61
Table3.Hostspecies,supergroupsandGenBankaccessionnumbersofcoxAandfbpAgenesforconstructing
thephylogenictree(Fig.2)ofWolbachiastrains.
Hostspecies Supergroup coxA fbpA
orsubspecies Accessionno. Accessionno.
Brugiamalayi D DQ842273 DQ842347
Cimexlectularius F DQ842275 DQ842349
Acromissparsa A DQ842271 DQ842345
Aedesalbopictus A DQ842268 DQ842342
Camponotuspennsylvanicus A DQ842276 DQ842350
Drosophilabifasciata A DQ842279 DQ842353
Drosophilainnubila A DQ842280 DQ842354
Drosophilamelanogaster A DQ842304 DQ842378
Drosophilaneotestacea A DQ842281 EU126408
Drosophilaorientacea A DQ842282 EU126398
Drosophilarecens A DQ842283 DQ842357
Ephestiakuehniella A DQ842289 DQ842363
Incisitermessnyderi A DQ842292 DQ842366
Muscidifuraxuniraptor A DQ842293 DQ842367
Nasoniagiraulti A DQ842294 DQ842368
Nasonialongicornis A DQ842295 DQ842369
Nasoniavitripennis A FJ390240 DQ842370
Solenopsisinvicta A DQ842300 DQ842374
Acraeaencedon B DQ842269 DQ842343
Acraeaeponina B DQ842270 DQ842344
Armadillidiumvulgare B FJ390241 EF451552
Chelymorphaalternans B DQ842274 DQ842348
Culexpipienspipiens B DQ842277 DQ842351
Culexpipiensquinquefasciatus B DQ842278 DQ842352
Drosophilasimulans B KF987018 KF987033
Encarsiaformosa B DQ842288 DQ842362
Gryllusfirmus B DQ842291 DQ842365
Nasoniavitripennis B DQ842297 DQ842371
Ostriniascapulalis B DQ842298 DQ842372
Protocalliphorasialia B DQ842299 DQ842373
Teleogryllustaiwanemma B DQ842303 DQ842377
Triboliumconfusum B DQ842301 DQ842375
Trichogrammadeion B DQ842302 DQ842376
coxADQ842273, fbpADQ842347) and Cimex fied:T.chilonis(Ishii,1941)(sevenpopulations),
lectularius (Latreille, 1802) (wsp DQ842459, T.evanescens(Westwood,1833)(sevenpopula-
coxADQ842275,fbpADQ842349). tions), T. ostriniae (Pang & Chen, 1974) (three
populations), T. embryophagum (Hartig, 1838)
(one population), T. dendrolimi (Matsumura,
3.Results 1926) (one population) and T. japonicum (Ash-
mead, 1904) (two populations). The ITS2 se-
3.1.Trichogrammaidentification quences obtained from each species were (97–
anddistribution 100% Max Ident score in BLAST) similar to
thosepresentinGenBank.Inthisstudy,atotalof
TheITS2sequencesthatwereobtainedfromeach eleven ITS2 sequences were deposited in Gen-
population were compared with the identified Bank (accession numbers KR148945–KR14-
ITS2sequencesofrDNAfromGenBanktocon- 8955)(Table1),andthesesequenceswerecom-
firmtheidentificationofTrichogrammaspecies pleteITS2sequencesplusflankingsequencesof
(Table4).SixTrichogrammaspecieswereidenti- 5.8Sand28S.Althoughthesesequencesdisplay
62 Liuetal. (cid:127) ENTOMOL.FENNICAVol.28
Table4.SequencesusedfromGenBankforcomparisonoftheTrichogrammaspeciesinthisstudy.
Sampleno. Sequence Basepairs Accessionno. Sequence
andspp. similarity(%)
1.T.chilonis partialITS-2 1,155 AY167415 99
2.T.chilonis partialITS-2 1,158 AY167418 99
3.T.chilonis CompleteITS-2 560 DQ088055 99
4.T.chilonis CompleteITS-2 538 GU562445 99
5.T.evanescens CompleteITS-2 559 DQ088059 99
6.T.evanescens CompleteITS-2 546 FJ436332 99
7.T.evanescens CompleteITS-2 546 JN315373 99
8.T.evanescens CompleteITS-2 546 JN315380 99
9.T.ostriniae CompleteITS-2 446 AY244463 98
10.T.ostriniae CompleteITS-2 447 AY518695 99
11.T.ostriniae CompleteITS-2 491 GQ324625 98
12.T.embryophagum CompleteITS-2 479 AF453562 100
13.T.embryophagum CompleteITS-2 474 AY244465 100
14.T.embryophagum CompleteITS-2 593 DQ344044 97
15.T.embryophagum CompleteITS-2 530 JF920430 97
16.T.dendrolimi partialITS-2 510 AB094398 98
17.T.dendrolimi CompleteITS-2 412 AF453555 99
18.T.dendrolimi CompleteITS-2 540 AF517576 98
19.T.dendrolimi CompleteITS-2 465 DQ344045 97
20.T.japonicum CompleteITS-2 578 DQ471294 98
21.T.japonicum partialITS-2 436 FN822756 98
22.T.japonicum partialITS-2 438 FN822758 98
23.T.japonicum partialITS-2 436 FN822759 98
significantinterspeciesdifferences,theyshowed TheT.ostriniaespecimensfromthreepopula-
low intraspecific variability in length (2–10 tionshadtwodifferentsequences:To–HBZand
bases). To–MZ (KR148945); To–HS (KR148946),
ThecomparisonshowedthatT.chiloniswas whichhadtwomutationsites(Table1).Thetwo
foundfromsevenpopulationswiththreedifferent sequences were closely similar (98–99%) to T.
sequences, Tc–CJ and Tc–WLP (KR148947); ostriniae in GenBank accession numbers
Tc–Lo, Tc–NB, Tc–MM, Tc–TW (KR148948); AY244463,AY518695andGQ324625(Table4).
Tc–HS (KR148949) (Table 1). The three se- The sequence of T. embryophagum, Tem–
quenceshadonlythreemutationsites,andthese- Tcac (KR148953), was closely similar (97–
quenceswerecloselysimilar(99%)toT.chilonis 100%) to GenBank accession numbers AF-
in GenBank accession numbers AY167415, 453562, AY244465, DQ344044 and JF920430
AY167418, DQ088055 and GU562445 (Table (Table4).
4). The sequence of T. dendrolimi (Td–NJ,
TheT.evanescensspecimensfromsevenpop- KR148954)wascloselysimilar(97–99%)tothat
ulations had three different sequences: Te–HS, of T. dendrolimi available in GenBank (AB-
Te–BJ,Te–CQ,Te–K1andTe–K2(KR148950); 094398,AF453555,AF517576andDQ344045)
Te–Tconf (KR148951); Te–GD (KR148952) (Table4).
(Table1).Thethreesequenceshad10mutation The sequence of T. japonicum (Tj–GD and
sites, and the sequences were closely similar Tj–HSKR148955)wascloselysimilar(98%)to
(99%) to T. evanescens in GenBank accession thatofT.japonicumavailableinGenBank(DQ-
numbers DQ088059, FJ436332, JN315373 and 471294,FN822756,FN822758andFN822759)
JN315380(Table4). (Table4).
ENTOMOL.FENNICAVol.28 (cid:127) MolecularidentificationofTrichogramma 63
3.2.Identificationoftheassociated
Wolbachiastrain
The detection of Wolbachia was performed for
Trichogrammaspeciesfrom21sites.Theinfec-
tionexistedinonlyfivepopulations,whichwere
Tc-TW,Te-BJ,Te-K1,Tem-TcacandTj-GD,and
all tested individuals of these five populations
wereinfected.Furthermore,allofthefivepopu-
lationswerefoundtohaveidenticalsequencesof
thewsp,coxAandfbpAgenes.Asthesefivepop-
ulationswereinfectedwiththesameWolbachia
strain,onlyoneofthem(Trichogrammajaponi-
cum) was used for the phylogenetic analysis
basedonwsp,coxAandfbpAgenes.
Thephylogeneticanalysiswasperformedfor
theTrichogrammaWolbachiawspsequencesand
29referencesequences.Thetreehastwomajor
branches,correspondingtosupergroupsAandB
(Fig. 1). The wsp sequence in Trichogramma
samplesshared100%identitywiththesequences
fromT.evanescens(AY390279).ThisWolbachia
strain was defined in the strain wEvaA in the
group of Eva of the supergroup A(Fig. 1). The
phylogenetictreefortheconcatenatedsequences
ofWolbachiacoxAandfbpAisshowninFig.2.
Similar to the wsp tree, the concatenated se-
quences were first clustered into supergroup A
branch. The concatenated sequences were clus-
tered into a subclade with Aedes albopictus
(Skuse, 1894) (coxA DQ842268 and fbpA
DQ842342),sharing99%identity(Fig.2).Asthe
topologies of the trees inferred from neighbor
joining and maximum parsimony were similar
based on wsp and concatenated coxAand fbpA
sequences, we only displayed the trees con-
structedbyneighborjoining(Figs1and2).The
sequencesforthewsp,fbpAandcoxAgeneswere
deposited in GenBank with the accession num-
Fig.1.Phylogeneticneighborjoiningtreeof bersKR906068,KR906069andKR906070.
Wolbachiabasedonwspsequences.Forinformation
ofwspgene,seeTable2.SequencesofBrugia
malayiandCimexlectulariuswereusedasoutgroups. 4.Discussion
Namesofhostspeciesandtheirrelatedgroupsare
listedontherightsideofthefigure.Supergroupsof
ITS2providesanexcellentmethodforseparating
Wolbachiaareshownasuppercaseletters.Thein-
closely related species of Trichogramma. The
fectedstrainofTrichogrammajaponicumclusteredto-
mainadvantageoftheDNAidentificationsystem
getherwiththatofTrichogrammaevanescens,which
overthemorphologicalsystemisthatitisfastand
werethestrainEva.Theyarehighlightedbyshaded
area.Bootstrapvalues>50%areshownabove requiresfewspecializedskills,andcanworkwell
branches. onthedriedor100%alcoholstoredspecimen(s).
64 Liuetal. (cid:127) ENTOMOL.FENNICAVol.28
Fig.2.PhylogeneticneighborjoiningtreeofWol-
bachiabasedonconcatenatedsequencesofcoxA
andfbpA.ForinformationofcoxAandfbpAgenes,
seeTable3.SequencesofBrugiamalayiandCimex
lectulariuswereusedasoutgroups.Namesofhost
speciesandtheirrelatedgroupsarelistedontheright
sideofthefigure.SupergroupsofWolbachiaare
shownasuppercaseletters.PopulationofTricho-
grammajaponicumishighlightedbyboldfaceinthe
figure.Bootstrapvalues>50%areshownabove
branches.
informationfortheidentificationofTrichogram-
maspecies.
In this study, the sequences of the tested
Trichogramma samples were blasted in NCBI
andthespecieswith98–100%identitywereiden-
tifiedasthesamespecies.Finally,theidentifica-
tion results of molecular methods and morpho-
logical characters were identical. Therefore, it
canbededucedthattheresultsofthemolecular
identificationbasedonITS2genesarereliable.
ThephylogenyofWolbachiahasbeenstudied
extensively based on different gene sequences
(Roussetetal.1992,Braigetal.1998).Astheex-
tensiverecombinationandstrongdiversifyingse-
lectionaffectthewspgene,itisanunreliabletool
forthecharacterizationofWolbachia(Werren&
Bartos 2001, Baldo et al. 2002, Jiggins et al.
2002,Baldoetal.2005).Therefore,anothertwo
Wolbachia genes, coxA and fbpA, were also
applied.
Inthisstudy,Wolbachiainfectionwasfound
only infivepopulationsofTrichogrammafrom
21 sites. The phylogenetic analysis of the Wol-
bachia wsp sequences revealed that the Wol-
bachiastrainbelongedtothestrainwEvaAinthe
groupofEvaofthesupergroupA.Thephylogen-
etictreeofalsotheconcatenatedcoxAandfbpA
revealed that the Wolbachia strain of Tricho-
grammawasdefinedassupergroupA,anditwas
closely, with 83% identity, related to the Wol-
bachiaofAedesalbopictus.
Inarecentstudyusingthewspgeneandfive
genes in multilocus sequence typing (MLST)
(Poorjavadetal.2012),onlytwopopulationsof
MoreandmoreresourcesofTrichogrammaITS2 Trichogrammabrassicae(Bezdenko,1968)from
genes are becoming publicly available from 34 tested Trichogramma populations were in-
NCBIdatabase,whichprovidesarichsourceof fectedbyWolbachia.Thetwopopulationswere
ENTOMOL.FENNICAVol.28 (cid:127) MolecularidentificationofTrichogramma 65
infected with the same Wolbachia strain, which Guo,Z.,Ruan,C.C.,Zang,L.S.,Zhang,F.&Jin,F.Y.
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ashmead, an egg parasitoid of Chilo suppressalis
AlthoughtheWolbachiainfectionrateswere
(Walker) in Jilin, China. — Biological Control 27:
low in both our current study and in that of
276–279.
Poorjavad et al. (2012), the Wolbachia strains Harris,H.L.,Braig,H.R.&Journal,A.2003:Spermchro-
weredifferent.ThetwoT.brassicaepopulations matinremodellingandWolbachia-inducedcytoplas-
were from Iran, i.e. Western Asia, whereas the micincompatibilityinDrosophila.—Biochemistry
andCellBiology81:229–240.
fiveinfectedpopulationsinourstudywerefrom
Hassan, S. A. 1994: Strategies to select Trichogramma
EastAsiaandSouth-EastAsia.Itisthuspossible
speciesforuseinbiologicalcontrol.–In:Wajnberg,E.
that the Trichogramma populations in Western &Hassan,S.A.(eds.),Biologicalcontrolwitheggpa-
AsiaareinfectedwithdifferentWolbachiastrains rasitoids: 55–71. CAB International, Wallingford
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The molecular identification and Wolbachia
thelytokycausingWolbachiainthepre-adultandadult
infectiontestfortheTrichogrammaspeciesfrom
life stages influence the adult fecundity of Tricho-
21populationscanprovideanimportantaidfor grammadeionandMuscidifuraxuniraptor?—Pro-
biologicalcontrolprogramsusingTrichogramma ceedings Experimental and Applied Entomology 6:
spp. 35–40.
Jiggins,F.M.,Hurst,G.D.D.&Yang,Z.H.2002:Host-
symbiont conflicts: positive selection on an outer
Acknowledgments. We thank two anonymous reviewers
membrane protein of parasitic but not mutualistic
fortheirvaluablecommentstoimprovethemanuscript.
Rickettsiaceae.—MolecularBiologyandEvolution
WealsothankProf.HongyingHufromXinjiangUniver-
19:1341–1349.
sityforTrichogrammamorphologicalidentification.This
Ko,K.,Liu,Y.D.,Hou,M.L.,Babendreier,D.,Zhang,F.
research was funded by the National Natural Science
& Song, K. 2014: Evaluation for potential Tricho-
FoundationofChina(31370439)andaEuropeAidproject
gramma (Hymenoptera: Trichogrammatidae) strains
(DCIFood/2010/230–238).
for control of the Striped Stem Borer (Lepidoptera:
Crambidae) in the Greater Mekong Subregion. —
JournalofEconomicEntomology107:955–963.
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