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Development of a Multiplex Method to Discriminate BetweenNeochrysocharis formosa (Hymenoptera Eulophidae)Reproductive ModesAuthor(s) Tetsuya Adachi-Hagimori and Kazuki MiuraSource Journal of Economic Entomology 101(4)1510-1514 2008Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-0493(2008)101[1510DOAMMT]20CO2URL httpwwwbiooneorgdoifull1016030022-0493282008291015B15103ADOAMMT5D20CO3B2

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SHORT COMMUNICATIONS

Development of a Multiplex Method to Discriminate BetweenNeochrysocharis formosa (Hymenoptera Eulophidae)

Reproductive Modes

TETSUYA ADACHI-HAGIMORI123 AND KAZUKI MIURA14

J Econ Entomol 101(4) 1510ETH1514 (2008)

ABSTRACT A multiplex polymerase chain reaction (PCR) method was applied to differentiatethelytokous and arrhenotokous strains of Neochrysocharis formosa (Westwood) Alignment of strainTHORNrst internal transcribed spacer regions revealed high nucleotide variability and the strain-speciTHORNcprimer sequence used Strains were easily differentiated after gel electrophoresis of multiplex PCRproductsbecausearrhenotokous specimensproduceda500-bp fragmentaswell as the800-bp fragmentcommon to both strains This method successfully distinguished N formosa strains regardless ofcollection site across Japan thus it is probably suitable for similar applications in Turkey Italy andelsewhere

KEYWORDS Neochrysocharis formosa Liriomyza trifolii Liriomyza sativae biological agent mul-tiplex polymerase chain reaction

The eulophid parasitoid Neochrysocharis formosa(Westwood) is one of the most important naturalenemies of leafminers Liriomyza trifolii (Burgess)and Liriomyza sativae Blanchard in Japan (Saito et al1996 Hondo et al 2006 Tokumaru and Abe 2006) Lir-iomyza huidobrensis (Blanchard) in Turkey (Civelek etal 2002) and agromyzids in Italy (Burgio et al 2007)The thelytokous strain of N formosa is a registeredbiological control agent in Japan (Tagami et al 2006)where both arrhenotokous and thelytokous strainshave been observed in sympatry (Arakaki and Kinjo1998 Maryana 2000) Thelytokous N formosa areknown to be infected by a parthenogenesis-inducingRickettsia bacterium (Hagimori et al 2006)

Life history and behavioral characteristics of the-lytokous parasitoid species infected by microorgan-isms are sometimes different to those of arrhenotok-ous strains (eg Wang and Smith 1996 Miura andTagami 2004) To effectively use N formosa in bio-logical control we need to estimate strain suitabilityby examining their respective characteristics includ-ing population distribution and preferred habitats Be-cause females from both strains are morphologicallyindistinguishable they must currently be differenti-ated by assessing offspring gender from ovipositing

virgin females a relatively cumbersome and time-consuming taskRickettsia infection status can be usedto discriminate strains collected in open agriculturalenvironments However because the thelytokousstrain will be used in greenhouses at relatively hightemperatures (30C during summer) Rickettsia in-fection may be thermally depleted or cured after re-lease as is known to happen withWolbachia-infectedwasps Under these circumstances nuclear or mito-chondrial primers would prove more robust Thus wedeveloped a simple one-step multiplex polymerasechain reaction (PCR) technique to facilitate rapididentiTHORNcation of N formosa strains during continuingbiological control studies

Species identity can be readily determined usingDNA sequence data from any of several mitochondrialand nuclear genes (Scheffer 2000 Scheffer and Lewis2001) but this method can be equally time-consumingand relatively expensive for those not routinely in-volved with DNA sequencing Multiplex PCR can pro-vide simultaneous ampliTHORNcation of several DNA frag-ments thus it is routinely applied to differentiatespecies in single PCR and gel electrophoresis steps(Hinomoto et al 2004) This method has been suc-cessfully developed for identiTHORNcation of insectssuch as rootworms (Roehrdanz 2003)Orius species(Hinomoto et al 2004) leafminers (Miura et al2004) and minute parasitoids (Davies et al 2006)

The THORNrst internal transcribed spacer (ITS-1) regionis part of the eukaryotic cistron of ribosomal DNAlocated between the genes coding for 18S and 58SrRNA (Schulenburg et al 2001) The ITS-1 spacer hasbecome a popular marker for phylogenetic studies ofinter- and intraspecies relatedness because it dem-

1 Graduate School of Biosphere Sciences Hiroshima UniversityHigashi-Hiroshima Hiroshima 739-8511 Japan

2 Research Fellow of the Japan Society for the Promotion of Sci-ence

3 Corresponding author and current address Hiroshima University6-12-1 Nishifukatsu Fukuyama 721-8514 Japan (e-mail tetsuya3affrcgojp)

4 National Agricultural Research Center for Western RegionFukuyama Hiroshima 721-8514 Japan

0022-0493081510ETH1514$04000 2008 Entomological Society of America

onstrates a comparatively high rate of evolution dueto its noncoding structure and it can be easily isolatedvia PCR from almost any taxon by using conservedprimers in adjacent coding regions (Hillis and Dixon1991 Bakker et al 1995 Buckler and Holtsford 1996Gouliamova and Hennebert 1998)

We investigated genetic variation in the ITS-1 re-gion of arrhenotokous and thelytokous N formosastrains by DNA sequencing and then we developed amultiplex PCR method to differentiate the two strains

Materials and Methods

Collection and preservation of the N formosa sam-ples Eggs and larvae of N formosa that parasitizedLiriomyza spp or Chromatomyia horticola (Goureau)were collected from Japan during 2005 2006 and 2007(collection details are shown in Table 1) All collectedhosts were incubated at 24C under a photoperiod of168 (LD) h until the emergence of wasps from thehost leafminers Parasitoid cultures were then initi-ated and maintained under the same environmentalconditions on L sativae mining cut leaves of kidneybeans Phaseolus vulgaris L maintained in conicalcylinders containing fresh water Thelytokous and ar-rhenotokous strains were differentiated by assessingoffspring gender from ovipositing virgin femalesDNAExtraction PCR andDNA SequencingDNA

was extracted from individual specimens by crushingthem singly with a clean plastic rod in 30 l of aTris-EDTA buffer (5 N NaCl 05 mM EDTA pH 80and 1 M Tris-HCl pH 80) and then incubating with2 l of 05 mg ml proteinase K at 37C for 05 hHomogenates were boiled at 999C for 3 min to in-activate proteinase K and then they were used astemplates for PCR

The ITS-1 regions of selected specimens were am-pliTHORNed using 18S 1975 F (5-TAACAAGGTTTCCG-TAGGTG-3) and 58S 35R (5-AGCTGGCTGCGT-TCTTCATCGA-3) primers (following Ratcliffe et al2002) in 33-l reactions containing 15 U of Taq poly-merase (Applied Biosystems Foster City CA) 066 lof 10 mM dNTPs each 13 l of 10 pmol l1 forwardand reverse primers 33 l of 10 PCR buffer withMgCl2 1 l of DNA template and 262 l of sterilewater PCR ampliTHORNcations were done in an ABI ther-mal cycler (PCR System 9700 Applied Biosystems)with the following program initial denaturing at 92C

for 1min 35 cycles of 92C for 1 min annealing at 58Cfor 1min then 72C for 1min 30 s and a THORNnal extensionstep of 72C for 1 min 30 s All PCRs included anegative control (sterile water instead of DNA) todetect DNA contamination PCR products were re-solved on 15 agarose gels stained with ethidiumbromide and visualized under an UV transilluminator

PCR products that yielded amplicons of the ex-pected size (800 bp) were cloned with p-GEMTEasy Vector system (Promega Tokyo Japan) Colo-nies containing the fragment of interest were isolatedpuriTHORNed and directly sequenced using M13M4 (5-GTTTTCCCAGTCACGAC-3) and M13RV (5-CA-GGAAACAGCTATGAC-3) universal sequencingprimers A dye terminator-labeled cycle sequencingreaction was conducted with BigDye DNA sequenc-ing kit version 31 (Applied Biosystems Tokyo Ja-pan) The temperature proTHORNle was 1 min at 96C fol-lowed by 25 cycles of 10 s at 96C 5 s at 50C and 4 minat 60C Reaction products were analyzed using anABI PRISM 3130xl genetic analyzer (Applied Biosys-tems) Partial sequences were edited and assembledwith Contig Express in Vector NTI Advance version101 (Invitrogen InforMax Frederick MD)

Multiple sequence alignments were performed us-ing Clustal W (Thompson et al 1994) and a speciTHORNcprimer for the arrhenotokous strain (NFARF 5-CCCCTGTGGCTGTACATCAT-3) was designed us-ing Primer 3 software (Rozen and Skaletsky 2000)

To check NFARF primer reliability we performeda multiplex PCR using AR-SHIZU TH-SHIZU AR-KYO TH-KYO AR-HIRO TH-HIRO AR-KAG andTH-KAG samples (Table 1) DNA isolation and mul-tiplex PCR were performed using the method de-scribed previously but with 13 l of 10 pmollNFARF and a reduced sterile water volume (249 l)in each PCR

Results and Discussion

ITS-1 regions of thelytokous and arrhenotokous Nformosa strains were determined (GenBank accessionnos AB363934 and AB363935 respectively) Align-ment of ITS-1 regions from both strains revealed 126of nucleotides varied between the two (753864-bpidentity) (Fig 1) The number of substitutions isgreater than indels (68 and 43 bp respectively) asfound in Drosophila species (Halligan et al 2004)DNA sequence and ampliTHORNed products showing vari-ation determined the gene regions most useful forsystematic differentiation Relatively high nucleotidedivergence suggests Rickettsia infection history in thethelytokous strain is relatively old and no or limitedgene szligow has occurred between strains Actual in-ter-strain gene szligow could be assessed in the futurewith crossing experiments that use nuclear primersdeveloped in this paper to check the inheritance ofparental genes

Arrhenotokous and thelytokous strains were eas-ily differentiated after gel electrophoresis of mul-tiplex PCR products because arrhenotokous spec-imens produced a 500-bp fragment along with the

Table 1 Collection details for N formosa specimens

Abbreviation City PrefectureHostszligya

Reproductivemodeb

Collectiontime

AR-SHI Fuji Shizuoka L AR May 2007TH-SHI Fuji Shizuoka L TH May 2007AR-KYO Nagaokakyo Kyoto L AR June 2005TH-KYO Nagaokakyo Kyoto L TH Oct 2005AR-HIRO Fukuyama Hiroshima C AR April 2007TH-HIRO Fukuyama Hiroshima C TH April 2005AR-KAG Takamatsu Kagawa C AR May 2006TH-KAG Takamatsu Kagawa C TH May 2006

a L Liriomyza spp C Chromatomyia horticolab AR arrhenotoky TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1511

Fig 1 Alignment N formosa ITS-1 sequences Underlines indicate primers used for multiplex PCR Primers 18S1975 F5-TAACAAGGTTTCCGTAGGTG-3 58S35R 5-AGCTGGCTGCGTTCTTCATCGA-3 NFARF 5-CCCCTGTGGCTG-TACATCAT-3 Asterisks represent residues identical to those of the reference sequence and dashes indicate alignment gapsBase substitutions are indicated by respective bases

1512 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

800-bp fragment common to both strains (Fig 2)Because the method readily distinguished strainsfrom geographically separate regions of Japan webelieve it may be suitable for discerning arrheno-tokous and thelytokous N formosa strains in othercountries where this important biological controlagent is applied

In Japan biological control using the thelytokousstrain of N formosa has only recently commencedLittle is currently known about the relative distri-bution preferred habitats and life history charac-teristics of both N formosa strains This multiplexPCR identiTHORNcation method will facilitate furtherresearch investigating these characteristics and soimprove biological control efTHORNcacy by using N for-mosa

Acknowledgments

We thank C Hagimori and Sumitomo Chemical Co Ltdfor collecting and providingN formosa samples respectivelyWethankAndrewPDavies forcritical readingof theoriginalmanuscript This research is partially supported by grant-in-aid for Japan Society for the Promotion of Science fellows (toTA-H)

References Cited

Arakaki N and K Kinjo 1998 Notes on the parasitoidfauna of the serpentine leafminer Liriomyza trifolii (Bur-gess) (Diptera Agromyzidae) in Okinawa southern Ja-pan Appl Entomol Zool 33 577ETH581

Bakker F T J L Olsen and W T Stam 1995 Evolutionof nuclear rDNA ITS sequences in the Cladophoraalbidasericea clade (Chlorophyta) J Mol Evol 40 640ETH651

Buckler E S and T P Holtsford 1996 Zea systematicsribosomal ITS evidence Mol Biol Evol 13 612ETH622

Burgio G A Lanzoni P Navone K VanAchterberg andAMasetti 2007 Parasitic hymenoptera fauna on Agromy-zidae (Diptera) colonizing weeds in ecological compen-sation areas in northern Italian agroecosystems J EconEntomol 100 298ETH306

Civelek H S Z Yoldas and P G Weintraub 2002 Theparasitoid complex of Liriomyza huidobrensis in cucum-ber greenhouses in Izmir Province western Turkey Phy-toparasitica 30 285ETH287

Davies A P C L Lange and S L OrsquoNeill 2006 A rapidsingle-step multiplex method for discriminating betweenTrichogramma (Hymenoptera Trichogrammatidae) spe-cies in Australia J Econ Entomol 99 2142ETH2145

Gouliamova D E and G L Hennebert 1998 Phyloge-netic relationships in the Saccharomyces cerevisiae com-plex of species Mycotaxon 66 337ETH353

Hagimori T Y Abe S Date and K Miura 2006 The THORNrstTHORNnding of a Rickettsia bacterium associated with parthe-nogenesis induction among insects Curr Microbiol 5297ETH101

Halligan D L A Eyre-Walker P Andolfatto and P DKeightley 2004 Patterns of evolutionary constraints inintronic and intergenic DNA ofDrosophilaGenome Res14 273ETH279

Hillis D M and M T Dixon 1991 Ribosomal DNA mo-lecular evolution and phylogenetic inference Q RevBiol 66 411ETH454

Hinomoto N M Muraji T Noda T Shimizu and KKawasaki 2004 IdentiTHORNcation of THORNve Orius species inJapan by multiplex polymerase chain reaction Biol Con-trol 31 276ETH279

Hondo T A Koike and T Sugimoto 2006 Comparison ofthermal tolerance of seven native species of parasitoids(Hymenoptera Eulophidae) as biological control agentsagainst Liriomyza trifolii (Diptera Agromyzidae) inJapan Appl Entomol Zool 41 73ETH82

Maryana N 2000 Studies on the ecological aspects of Neo-chrysocharis formosa(Hymenoptera Eulophidae) attack-ing Liriomyza trifolii (Diptera Agromyzidae) PhD dis-sertation Kyusyu University Fukuoka Japan

Miura K and Y Tagami 2004 Comparison of life historycharacters of arrhenotokous andWolbachia-associated the-lytokous Trichogramma kaykai Pinto and Stouthamer (Hy-menoptera Trichogrammatidae) Ann Entomol Soc Am97 765ETH769

Miura K Y Tagami M Ohtaishi and A Iwasaki 2004Application of molecular techniques to distinguishLiriomyza trifolii fromL sativae (Diptera Agromyzidae)on tomato cultivation in Japan J Econ Entomol 97964ETH969

Ratcliffe S T H M Robertson C J Jones G A Bolleroand R A Weinzierl 2002 Assessment of parasitism ofhouse szligy and stable szligy (Diptera Muscidae) pupae bypteromalid (Hymenoptera Pteromalidae) parasitoids us-ing a polymerase chain reaction assay J Med Entomol39 52ETH60

RoehrdanzRL 2003 Multiplexpolymerasechain reactionmethod for differentiating western and northern cornrootworm larvae (Coleoptera Chrysomelidae) J EconEntomol 96 669ETH672

Rozen S andH Skaletsky 2000 Primer3 on the WWW forgeneral users and for biologist programmers MethodsMol Biol 132 365ETH386

Saito T F Ikeda and A Ozawa 1996 Effect of pesticideson parasitoid complex of serpentine leafminer Liriomyzatrifolii (Burgess) in Shizuoka Prefecture Jpn J ApplEntomol Zool 40 127ETH133

Scheffer S J 2000 Molecular evidence of cryptic specieswithin the Liriomyza huidobrensis (Diptera Agromyzi-dae) J Econ Entomol 93 1146ETH1151

Scheffer S J and M L Lewis 2001 Two nuclear genesconTHORNrm mitochondrial evidence of cryptic species withinLiriomyza huidobrensis (Diptera Agromyzidae) AnnEntomol Soc Am 94 648ETH653

Schulenburg J J M Hancock A Pagnamenta J J SloggettMEN Majerus and GDD Hurst 2001 Extremelength and length variation in the THORNrst ribosomal internal

Fig 2 Banding patterns obtained by gel electrophoresisafter multiplex PCR ofN formosa adults AR arrhenotoky M100-bp DNA ladder (MBI Fermentas Hanover MD) SWsterile water negative control TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1513

transcribed spacer of ladybird beetles (Coleoptera Coc-cinellidae) Mol Biol Evol 18 648ETH660

Tagami Y M Doi K Sugiyama A Tatara and T Saito2006 Survey of leafminers and their parasitoids to THORNndendosymbionts for improvement of biological controlBiol Control 38 210ETH216

Thompson J D D G Higgins and T J Gibson 1994Clustal W improving the sensitivity of progressive mul-tiple sequence alignment through sequence weightingposition speciTHORNc gap penalties and weight matrix choiceNucleic Acids Res 22 4673ETH4680

Tokumaru S and Y Abe 2006 Hymenopterous parasi-toids of leafminersLiriomyza sativaeBlanchardL trifolii(Burgess) and L bryoniae (Kaltenbach) in Kyoto pre-fecture Jpn J Appl Entomol Zool 50 341ETH345

Wang Z Y and S M Smith 1996 Phenotypic differencesbetween thelytokous and arrhenotokous Trichogrammaminutum fromZeiraphera canadensisEntomol Exp Appl78 315ETH323

Received 21 December 2007 accepted 24 February 2008

1514 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

onstrates a comparatively high rate of evolution dueto its noncoding structure and it can be easily isolatedvia PCR from almost any taxon by using conservedprimers in adjacent coding regions (Hillis and Dixon1991 Bakker et al 1995 Buckler and Holtsford 1996Gouliamova and Hennebert 1998)

We investigated genetic variation in the ITS-1 re-gion of arrhenotokous and thelytokous N formosastrains by DNA sequencing and then we developed amultiplex PCR method to differentiate the two strains

Materials and Methods

Collection and preservation of the N formosa sam-ples Eggs and larvae of N formosa that parasitizedLiriomyza spp or Chromatomyia horticola (Goureau)were collected from Japan during 2005 2006 and 2007(collection details are shown in Table 1) All collectedhosts were incubated at 24C under a photoperiod of168 (LD) h until the emergence of wasps from thehost leafminers Parasitoid cultures were then initi-ated and maintained under the same environmentalconditions on L sativae mining cut leaves of kidneybeans Phaseolus vulgaris L maintained in conicalcylinders containing fresh water Thelytokous and ar-rhenotokous strains were differentiated by assessingoffspring gender from ovipositing virgin femalesDNAExtraction PCR andDNA SequencingDNA

was extracted from individual specimens by crushingthem singly with a clean plastic rod in 30 l of aTris-EDTA buffer (5 N NaCl 05 mM EDTA pH 80and 1 M Tris-HCl pH 80) and then incubating with2 l of 05 mg ml proteinase K at 37C for 05 hHomogenates were boiled at 999C for 3 min to in-activate proteinase K and then they were used astemplates for PCR

The ITS-1 regions of selected specimens were am-pliTHORNed using 18S 1975 F (5-TAACAAGGTTTCCG-TAGGTG-3) and 58S 35R (5-AGCTGGCTGCGT-TCTTCATCGA-3) primers (following Ratcliffe et al2002) in 33-l reactions containing 15 U of Taq poly-merase (Applied Biosystems Foster City CA) 066 lof 10 mM dNTPs each 13 l of 10 pmol l1 forwardand reverse primers 33 l of 10 PCR buffer withMgCl2 1 l of DNA template and 262 l of sterilewater PCR ampliTHORNcations were done in an ABI ther-mal cycler (PCR System 9700 Applied Biosystems)with the following program initial denaturing at 92C

for 1min 35 cycles of 92C for 1 min annealing at 58Cfor 1min then 72C for 1min 30 s and a THORNnal extensionstep of 72C for 1 min 30 s All PCRs included anegative control (sterile water instead of DNA) todetect DNA contamination PCR products were re-solved on 15 agarose gels stained with ethidiumbromide and visualized under an UV transilluminator

PCR products that yielded amplicons of the ex-pected size (800 bp) were cloned with p-GEMTEasy Vector system (Promega Tokyo Japan) Colo-nies containing the fragment of interest were isolatedpuriTHORNed and directly sequenced using M13M4 (5-GTTTTCCCAGTCACGAC-3) and M13RV (5-CA-GGAAACAGCTATGAC-3) universal sequencingprimers A dye terminator-labeled cycle sequencingreaction was conducted with BigDye DNA sequenc-ing kit version 31 (Applied Biosystems Tokyo Ja-pan) The temperature proTHORNle was 1 min at 96C fol-lowed by 25 cycles of 10 s at 96C 5 s at 50C and 4 minat 60C Reaction products were analyzed using anABI PRISM 3130xl genetic analyzer (Applied Biosys-tems) Partial sequences were edited and assembledwith Contig Express in Vector NTI Advance version101 (Invitrogen InforMax Frederick MD)

Multiple sequence alignments were performed us-ing Clustal W (Thompson et al 1994) and a speciTHORNcprimer for the arrhenotokous strain (NFARF 5-CCCCTGTGGCTGTACATCAT-3) was designed us-ing Primer 3 software (Rozen and Skaletsky 2000)

To check NFARF primer reliability we performeda multiplex PCR using AR-SHIZU TH-SHIZU AR-KYO TH-KYO AR-HIRO TH-HIRO AR-KAG andTH-KAG samples (Table 1) DNA isolation and mul-tiplex PCR were performed using the method de-scribed previously but with 13 l of 10 pmollNFARF and a reduced sterile water volume (249 l)in each PCR

Results and Discussion

ITS-1 regions of thelytokous and arrhenotokous Nformosa strains were determined (GenBank accessionnos AB363934 and AB363935 respectively) Align-ment of ITS-1 regions from both strains revealed 126of nucleotides varied between the two (753864-bpidentity) (Fig 1) The number of substitutions isgreater than indels (68 and 43 bp respectively) asfound in Drosophila species (Halligan et al 2004)DNA sequence and ampliTHORNed products showing vari-ation determined the gene regions most useful forsystematic differentiation Relatively high nucleotidedivergence suggests Rickettsia infection history in thethelytokous strain is relatively old and no or limitedgene szligow has occurred between strains Actual in-ter-strain gene szligow could be assessed in the futurewith crossing experiments that use nuclear primersdeveloped in this paper to check the inheritance ofparental genes

Arrhenotokous and thelytokous strains were eas-ily differentiated after gel electrophoresis of mul-tiplex PCR products because arrhenotokous spec-imens produced a 500-bp fragment along with the

Table 1 Collection details for N formosa specimens

Abbreviation City PrefectureHostszligya

Reproductivemodeb

Collectiontime

AR-SHI Fuji Shizuoka L AR May 2007TH-SHI Fuji Shizuoka L TH May 2007AR-KYO Nagaokakyo Kyoto L AR June 2005TH-KYO Nagaokakyo Kyoto L TH Oct 2005AR-HIRO Fukuyama Hiroshima C AR April 2007TH-HIRO Fukuyama Hiroshima C TH April 2005AR-KAG Takamatsu Kagawa C AR May 2006TH-KAG Takamatsu Kagawa C TH May 2006

a L Liriomyza spp C Chromatomyia horticolab AR arrhenotoky TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1511

Fig 1 Alignment N formosa ITS-1 sequences Underlines indicate primers used for multiplex PCR Primers 18S1975 F5-TAACAAGGTTTCCGTAGGTG-3 58S35R 5-AGCTGGCTGCGTTCTTCATCGA-3 NFARF 5-CCCCTGTGGCTG-TACATCAT-3 Asterisks represent residues identical to those of the reference sequence and dashes indicate alignment gapsBase substitutions are indicated by respective bases

1512 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

800-bp fragment common to both strains (Fig 2)Because the method readily distinguished strainsfrom geographically separate regions of Japan webelieve it may be suitable for discerning arrheno-tokous and thelytokous N formosa strains in othercountries where this important biological controlagent is applied

In Japan biological control using the thelytokousstrain of N formosa has only recently commencedLittle is currently known about the relative distri-bution preferred habitats and life history charac-teristics of both N formosa strains This multiplexPCR identiTHORNcation method will facilitate furtherresearch investigating these characteristics and soimprove biological control efTHORNcacy by using N for-mosa

Acknowledgments

We thank C Hagimori and Sumitomo Chemical Co Ltdfor collecting and providingN formosa samples respectivelyWethankAndrewPDavies forcritical readingof theoriginalmanuscript This research is partially supported by grant-in-aid for Japan Society for the Promotion of Science fellows (toTA-H)

References Cited

Arakaki N and K Kinjo 1998 Notes on the parasitoidfauna of the serpentine leafminer Liriomyza trifolii (Bur-gess) (Diptera Agromyzidae) in Okinawa southern Ja-pan Appl Entomol Zool 33 577ETH581

Bakker F T J L Olsen and W T Stam 1995 Evolutionof nuclear rDNA ITS sequences in the Cladophoraalbidasericea clade (Chlorophyta) J Mol Evol 40 640ETH651

Buckler E S and T P Holtsford 1996 Zea systematicsribosomal ITS evidence Mol Biol Evol 13 612ETH622

Burgio G A Lanzoni P Navone K VanAchterberg andAMasetti 2007 Parasitic hymenoptera fauna on Agromy-zidae (Diptera) colonizing weeds in ecological compen-sation areas in northern Italian agroecosystems J EconEntomol 100 298ETH306

Civelek H S Z Yoldas and P G Weintraub 2002 Theparasitoid complex of Liriomyza huidobrensis in cucum-ber greenhouses in Izmir Province western Turkey Phy-toparasitica 30 285ETH287

Davies A P C L Lange and S L OrsquoNeill 2006 A rapidsingle-step multiplex method for discriminating betweenTrichogramma (Hymenoptera Trichogrammatidae) spe-cies in Australia J Econ Entomol 99 2142ETH2145

Gouliamova D E and G L Hennebert 1998 Phyloge-netic relationships in the Saccharomyces cerevisiae com-plex of species Mycotaxon 66 337ETH353

Hagimori T Y Abe S Date and K Miura 2006 The THORNrstTHORNnding of a Rickettsia bacterium associated with parthe-nogenesis induction among insects Curr Microbiol 5297ETH101

Halligan D L A Eyre-Walker P Andolfatto and P DKeightley 2004 Patterns of evolutionary constraints inintronic and intergenic DNA ofDrosophilaGenome Res14 273ETH279

Hillis D M and M T Dixon 1991 Ribosomal DNA mo-lecular evolution and phylogenetic inference Q RevBiol 66 411ETH454

Hinomoto N M Muraji T Noda T Shimizu and KKawasaki 2004 IdentiTHORNcation of THORNve Orius species inJapan by multiplex polymerase chain reaction Biol Con-trol 31 276ETH279

Hondo T A Koike and T Sugimoto 2006 Comparison ofthermal tolerance of seven native species of parasitoids(Hymenoptera Eulophidae) as biological control agentsagainst Liriomyza trifolii (Diptera Agromyzidae) inJapan Appl Entomol Zool 41 73ETH82

Maryana N 2000 Studies on the ecological aspects of Neo-chrysocharis formosa(Hymenoptera Eulophidae) attack-ing Liriomyza trifolii (Diptera Agromyzidae) PhD dis-sertation Kyusyu University Fukuoka Japan

Miura K and Y Tagami 2004 Comparison of life historycharacters of arrhenotokous andWolbachia-associated the-lytokous Trichogramma kaykai Pinto and Stouthamer (Hy-menoptera Trichogrammatidae) Ann Entomol Soc Am97 765ETH769

Miura K Y Tagami M Ohtaishi and A Iwasaki 2004Application of molecular techniques to distinguishLiriomyza trifolii fromL sativae (Diptera Agromyzidae)on tomato cultivation in Japan J Econ Entomol 97964ETH969

Ratcliffe S T H M Robertson C J Jones G A Bolleroand R A Weinzierl 2002 Assessment of parasitism ofhouse szligy and stable szligy (Diptera Muscidae) pupae bypteromalid (Hymenoptera Pteromalidae) parasitoids us-ing a polymerase chain reaction assay J Med Entomol39 52ETH60

RoehrdanzRL 2003 Multiplexpolymerasechain reactionmethod for differentiating western and northern cornrootworm larvae (Coleoptera Chrysomelidae) J EconEntomol 96 669ETH672

Rozen S andH Skaletsky 2000 Primer3 on the WWW forgeneral users and for biologist programmers MethodsMol Biol 132 365ETH386

Saito T F Ikeda and A Ozawa 1996 Effect of pesticideson parasitoid complex of serpentine leafminer Liriomyzatrifolii (Burgess) in Shizuoka Prefecture Jpn J ApplEntomol Zool 40 127ETH133

Scheffer S J 2000 Molecular evidence of cryptic specieswithin the Liriomyza huidobrensis (Diptera Agromyzi-dae) J Econ Entomol 93 1146ETH1151

Scheffer S J and M L Lewis 2001 Two nuclear genesconTHORNrm mitochondrial evidence of cryptic species withinLiriomyza huidobrensis (Diptera Agromyzidae) AnnEntomol Soc Am 94 648ETH653

Schulenburg J J M Hancock A Pagnamenta J J SloggettMEN Majerus and GDD Hurst 2001 Extremelength and length variation in the THORNrst ribosomal internal

Fig 2 Banding patterns obtained by gel electrophoresisafter multiplex PCR ofN formosa adults AR arrhenotoky M100-bp DNA ladder (MBI Fermentas Hanover MD) SWsterile water negative control TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1513

transcribed spacer of ladybird beetles (Coleoptera Coc-cinellidae) Mol Biol Evol 18 648ETH660

Tagami Y M Doi K Sugiyama A Tatara and T Saito2006 Survey of leafminers and their parasitoids to THORNndendosymbionts for improvement of biological controlBiol Control 38 210ETH216

Thompson J D D G Higgins and T J Gibson 1994Clustal W improving the sensitivity of progressive mul-tiple sequence alignment through sequence weightingposition speciTHORNc gap penalties and weight matrix choiceNucleic Acids Res 22 4673ETH4680

Tokumaru S and Y Abe 2006 Hymenopterous parasi-toids of leafminersLiriomyza sativaeBlanchardL trifolii(Burgess) and L bryoniae (Kaltenbach) in Kyoto pre-fecture Jpn J Appl Entomol Zool 50 341ETH345

Wang Z Y and S M Smith 1996 Phenotypic differencesbetween thelytokous and arrhenotokous Trichogrammaminutum fromZeiraphera canadensisEntomol Exp Appl78 315ETH323

Received 21 December 2007 accepted 24 February 2008

1514 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

Fig 1 Alignment N formosa ITS-1 sequences Underlines indicate primers used for multiplex PCR Primers 18S1975 F5-TAACAAGGTTTCCGTAGGTG-3 58S35R 5-AGCTGGCTGCGTTCTTCATCGA-3 NFARF 5-CCCCTGTGGCTG-TACATCAT-3 Asterisks represent residues identical to those of the reference sequence and dashes indicate alignment gapsBase substitutions are indicated by respective bases

1512 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

800-bp fragment common to both strains (Fig 2)Because the method readily distinguished strainsfrom geographically separate regions of Japan webelieve it may be suitable for discerning arrheno-tokous and thelytokous N formosa strains in othercountries where this important biological controlagent is applied

In Japan biological control using the thelytokousstrain of N formosa has only recently commencedLittle is currently known about the relative distri-bution preferred habitats and life history charac-teristics of both N formosa strains This multiplexPCR identiTHORNcation method will facilitate furtherresearch investigating these characteristics and soimprove biological control efTHORNcacy by using N for-mosa

Acknowledgments

We thank C Hagimori and Sumitomo Chemical Co Ltdfor collecting and providingN formosa samples respectivelyWethankAndrewPDavies forcritical readingof theoriginalmanuscript This research is partially supported by grant-in-aid for Japan Society for the Promotion of Science fellows (toTA-H)

References Cited

Arakaki N and K Kinjo 1998 Notes on the parasitoidfauna of the serpentine leafminer Liriomyza trifolii (Bur-gess) (Diptera Agromyzidae) in Okinawa southern Ja-pan Appl Entomol Zool 33 577ETH581

Bakker F T J L Olsen and W T Stam 1995 Evolutionof nuclear rDNA ITS sequences in the Cladophoraalbidasericea clade (Chlorophyta) J Mol Evol 40 640ETH651

Buckler E S and T P Holtsford 1996 Zea systematicsribosomal ITS evidence Mol Biol Evol 13 612ETH622

Burgio G A Lanzoni P Navone K VanAchterberg andAMasetti 2007 Parasitic hymenoptera fauna on Agromy-zidae (Diptera) colonizing weeds in ecological compen-sation areas in northern Italian agroecosystems J EconEntomol 100 298ETH306

Civelek H S Z Yoldas and P G Weintraub 2002 Theparasitoid complex of Liriomyza huidobrensis in cucum-ber greenhouses in Izmir Province western Turkey Phy-toparasitica 30 285ETH287

Davies A P C L Lange and S L OrsquoNeill 2006 A rapidsingle-step multiplex method for discriminating betweenTrichogramma (Hymenoptera Trichogrammatidae) spe-cies in Australia J Econ Entomol 99 2142ETH2145

Gouliamova D E and G L Hennebert 1998 Phyloge-netic relationships in the Saccharomyces cerevisiae com-plex of species Mycotaxon 66 337ETH353

Hagimori T Y Abe S Date and K Miura 2006 The THORNrstTHORNnding of a Rickettsia bacterium associated with parthe-nogenesis induction among insects Curr Microbiol 5297ETH101

Halligan D L A Eyre-Walker P Andolfatto and P DKeightley 2004 Patterns of evolutionary constraints inintronic and intergenic DNA ofDrosophilaGenome Res14 273ETH279

Hillis D M and M T Dixon 1991 Ribosomal DNA mo-lecular evolution and phylogenetic inference Q RevBiol 66 411ETH454

Hinomoto N M Muraji T Noda T Shimizu and KKawasaki 2004 IdentiTHORNcation of THORNve Orius species inJapan by multiplex polymerase chain reaction Biol Con-trol 31 276ETH279

Hondo T A Koike and T Sugimoto 2006 Comparison ofthermal tolerance of seven native species of parasitoids(Hymenoptera Eulophidae) as biological control agentsagainst Liriomyza trifolii (Diptera Agromyzidae) inJapan Appl Entomol Zool 41 73ETH82

Maryana N 2000 Studies on the ecological aspects of Neo-chrysocharis formosa(Hymenoptera Eulophidae) attack-ing Liriomyza trifolii (Diptera Agromyzidae) PhD dis-sertation Kyusyu University Fukuoka Japan

Miura K and Y Tagami 2004 Comparison of life historycharacters of arrhenotokous andWolbachia-associated the-lytokous Trichogramma kaykai Pinto and Stouthamer (Hy-menoptera Trichogrammatidae) Ann Entomol Soc Am97 765ETH769

Miura K Y Tagami M Ohtaishi and A Iwasaki 2004Application of molecular techniques to distinguishLiriomyza trifolii fromL sativae (Diptera Agromyzidae)on tomato cultivation in Japan J Econ Entomol 97964ETH969

Ratcliffe S T H M Robertson C J Jones G A Bolleroand R A Weinzierl 2002 Assessment of parasitism ofhouse szligy and stable szligy (Diptera Muscidae) pupae bypteromalid (Hymenoptera Pteromalidae) parasitoids us-ing a polymerase chain reaction assay J Med Entomol39 52ETH60

RoehrdanzRL 2003 Multiplexpolymerasechain reactionmethod for differentiating western and northern cornrootworm larvae (Coleoptera Chrysomelidae) J EconEntomol 96 669ETH672

Rozen S andH Skaletsky 2000 Primer3 on the WWW forgeneral users and for biologist programmers MethodsMol Biol 132 365ETH386

Saito T F Ikeda and A Ozawa 1996 Effect of pesticideson parasitoid complex of serpentine leafminer Liriomyzatrifolii (Burgess) in Shizuoka Prefecture Jpn J ApplEntomol Zool 40 127ETH133

Scheffer S J 2000 Molecular evidence of cryptic specieswithin the Liriomyza huidobrensis (Diptera Agromyzi-dae) J Econ Entomol 93 1146ETH1151

Scheffer S J and M L Lewis 2001 Two nuclear genesconTHORNrm mitochondrial evidence of cryptic species withinLiriomyza huidobrensis (Diptera Agromyzidae) AnnEntomol Soc Am 94 648ETH653

Schulenburg J J M Hancock A Pagnamenta J J SloggettMEN Majerus and GDD Hurst 2001 Extremelength and length variation in the THORNrst ribosomal internal

Fig 2 Banding patterns obtained by gel electrophoresisafter multiplex PCR ofN formosa adults AR arrhenotoky M100-bp DNA ladder (MBI Fermentas Hanover MD) SWsterile water negative control TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1513

transcribed spacer of ladybird beetles (Coleoptera Coc-cinellidae) Mol Biol Evol 18 648ETH660

Tagami Y M Doi K Sugiyama A Tatara and T Saito2006 Survey of leafminers and their parasitoids to THORNndendosymbionts for improvement of biological controlBiol Control 38 210ETH216

Thompson J D D G Higgins and T J Gibson 1994Clustal W improving the sensitivity of progressive mul-tiple sequence alignment through sequence weightingposition speciTHORNc gap penalties and weight matrix choiceNucleic Acids Res 22 4673ETH4680

Tokumaru S and Y Abe 2006 Hymenopterous parasi-toids of leafminersLiriomyza sativaeBlanchardL trifolii(Burgess) and L bryoniae (Kaltenbach) in Kyoto pre-fecture Jpn J Appl Entomol Zool 50 341ETH345

Wang Z Y and S M Smith 1996 Phenotypic differencesbetween thelytokous and arrhenotokous Trichogrammaminutum fromZeiraphera canadensisEntomol Exp Appl78 315ETH323

Received 21 December 2007 accepted 24 February 2008

1514 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

800-bp fragment common to both strains (Fig 2)Because the method readily distinguished strainsfrom geographically separate regions of Japan webelieve it may be suitable for discerning arrheno-tokous and thelytokous N formosa strains in othercountries where this important biological controlagent is applied

In Japan biological control using the thelytokousstrain of N formosa has only recently commencedLittle is currently known about the relative distri-bution preferred habitats and life history charac-teristics of both N formosa strains This multiplexPCR identiTHORNcation method will facilitate furtherresearch investigating these characteristics and soimprove biological control efTHORNcacy by using N for-mosa

Acknowledgments

We thank C Hagimori and Sumitomo Chemical Co Ltdfor collecting and providingN formosa samples respectivelyWethankAndrewPDavies forcritical readingof theoriginalmanuscript This research is partially supported by grant-in-aid for Japan Society for the Promotion of Science fellows (toTA-H)

References Cited

Arakaki N and K Kinjo 1998 Notes on the parasitoidfauna of the serpentine leafminer Liriomyza trifolii (Bur-gess) (Diptera Agromyzidae) in Okinawa southern Ja-pan Appl Entomol Zool 33 577ETH581

Bakker F T J L Olsen and W T Stam 1995 Evolutionof nuclear rDNA ITS sequences in the Cladophoraalbidasericea clade (Chlorophyta) J Mol Evol 40 640ETH651

Buckler E S and T P Holtsford 1996 Zea systematicsribosomal ITS evidence Mol Biol Evol 13 612ETH622

Burgio G A Lanzoni P Navone K VanAchterberg andAMasetti 2007 Parasitic hymenoptera fauna on Agromy-zidae (Diptera) colonizing weeds in ecological compen-sation areas in northern Italian agroecosystems J EconEntomol 100 298ETH306

Civelek H S Z Yoldas and P G Weintraub 2002 Theparasitoid complex of Liriomyza huidobrensis in cucum-ber greenhouses in Izmir Province western Turkey Phy-toparasitica 30 285ETH287

Davies A P C L Lange and S L OrsquoNeill 2006 A rapidsingle-step multiplex method for discriminating betweenTrichogramma (Hymenoptera Trichogrammatidae) spe-cies in Australia J Econ Entomol 99 2142ETH2145

Gouliamova D E and G L Hennebert 1998 Phyloge-netic relationships in the Saccharomyces cerevisiae com-plex of species Mycotaxon 66 337ETH353

Hagimori T Y Abe S Date and K Miura 2006 The THORNrstTHORNnding of a Rickettsia bacterium associated with parthe-nogenesis induction among insects Curr Microbiol 5297ETH101

Halligan D L A Eyre-Walker P Andolfatto and P DKeightley 2004 Patterns of evolutionary constraints inintronic and intergenic DNA ofDrosophilaGenome Res14 273ETH279

Hillis D M and M T Dixon 1991 Ribosomal DNA mo-lecular evolution and phylogenetic inference Q RevBiol 66 411ETH454

Hinomoto N M Muraji T Noda T Shimizu and KKawasaki 2004 IdentiTHORNcation of THORNve Orius species inJapan by multiplex polymerase chain reaction Biol Con-trol 31 276ETH279

Hondo T A Koike and T Sugimoto 2006 Comparison ofthermal tolerance of seven native species of parasitoids(Hymenoptera Eulophidae) as biological control agentsagainst Liriomyza trifolii (Diptera Agromyzidae) inJapan Appl Entomol Zool 41 73ETH82

Maryana N 2000 Studies on the ecological aspects of Neo-chrysocharis formosa(Hymenoptera Eulophidae) attack-ing Liriomyza trifolii (Diptera Agromyzidae) PhD dis-sertation Kyusyu University Fukuoka Japan

Miura K and Y Tagami 2004 Comparison of life historycharacters of arrhenotokous andWolbachia-associated the-lytokous Trichogramma kaykai Pinto and Stouthamer (Hy-menoptera Trichogrammatidae) Ann Entomol Soc Am97 765ETH769

Miura K Y Tagami M Ohtaishi and A Iwasaki 2004Application of molecular techniques to distinguishLiriomyza trifolii fromL sativae (Diptera Agromyzidae)on tomato cultivation in Japan J Econ Entomol 97964ETH969

Ratcliffe S T H M Robertson C J Jones G A Bolleroand R A Weinzierl 2002 Assessment of parasitism ofhouse szligy and stable szligy (Diptera Muscidae) pupae bypteromalid (Hymenoptera Pteromalidae) parasitoids us-ing a polymerase chain reaction assay J Med Entomol39 52ETH60

RoehrdanzRL 2003 Multiplexpolymerasechain reactionmethod for differentiating western and northern cornrootworm larvae (Coleoptera Chrysomelidae) J EconEntomol 96 669ETH672

Rozen S andH Skaletsky 2000 Primer3 on the WWW forgeneral users and for biologist programmers MethodsMol Biol 132 365ETH386

Saito T F Ikeda and A Ozawa 1996 Effect of pesticideson parasitoid complex of serpentine leafminer Liriomyzatrifolii (Burgess) in Shizuoka Prefecture Jpn J ApplEntomol Zool 40 127ETH133

Scheffer S J 2000 Molecular evidence of cryptic specieswithin the Liriomyza huidobrensis (Diptera Agromyzi-dae) J Econ Entomol 93 1146ETH1151

Scheffer S J and M L Lewis 2001 Two nuclear genesconTHORNrm mitochondrial evidence of cryptic species withinLiriomyza huidobrensis (Diptera Agromyzidae) AnnEntomol Soc Am 94 648ETH653

Schulenburg J J M Hancock A Pagnamenta J J SloggettMEN Majerus and GDD Hurst 2001 Extremelength and length variation in the THORNrst ribosomal internal

Fig 2 Banding patterns obtained by gel electrophoresisafter multiplex PCR ofN formosa adults AR arrhenotoky M100-bp DNA ladder (MBI Fermentas Hanover MD) SWsterile water negative control TH thelytoky

August 2008 ADACHI-HAGIMORI AND MIURA DIFFERENTIATION OF N formosa REPRODUCTIVE MODES 1513

transcribed spacer of ladybird beetles (Coleoptera Coc-cinellidae) Mol Biol Evol 18 648ETH660

Tagami Y M Doi K Sugiyama A Tatara and T Saito2006 Survey of leafminers and their parasitoids to THORNndendosymbionts for improvement of biological controlBiol Control 38 210ETH216

Thompson J D D G Higgins and T J Gibson 1994Clustal W improving the sensitivity of progressive mul-tiple sequence alignment through sequence weightingposition speciTHORNc gap penalties and weight matrix choiceNucleic Acids Res 22 4673ETH4680

Tokumaru S and Y Abe 2006 Hymenopterous parasi-toids of leafminersLiriomyza sativaeBlanchardL trifolii(Burgess) and L bryoniae (Kaltenbach) in Kyoto pre-fecture Jpn J Appl Entomol Zool 50 341ETH345

Wang Z Y and S M Smith 1996 Phenotypic differencesbetween thelytokous and arrhenotokous Trichogrammaminutum fromZeiraphera canadensisEntomol Exp Appl78 315ETH323

Received 21 December 2007 accepted 24 February 2008

1514 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4

transcribed spacer of ladybird beetles (Coleoptera Coc-cinellidae) Mol Biol Evol 18 648ETH660

Tagami Y M Doi K Sugiyama A Tatara and T Saito2006 Survey of leafminers and their parasitoids to THORNndendosymbionts for improvement of biological controlBiol Control 38 210ETH216

Thompson J D D G Higgins and T J Gibson 1994Clustal W improving the sensitivity of progressive mul-tiple sequence alignment through sequence weightingposition speciTHORNc gap penalties and weight matrix choiceNucleic Acids Res 22 4673ETH4680

Tokumaru S and Y Abe 2006 Hymenopterous parasi-toids of leafminersLiriomyza sativaeBlanchardL trifolii(Burgess) and L bryoniae (Kaltenbach) in Kyoto pre-fecture Jpn J Appl Entomol Zool 50 341ETH345

Wang Z Y and S M Smith 1996 Phenotypic differencesbetween thelytokous and arrhenotokous Trichogrammaminutum fromZeiraphera canadensisEntomol Exp Appl78 315ETH323

Received 21 December 2007 accepted 24 February 2008

1514 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 101 no 4