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Determining Parasitoid Species Composition in a Host Population: A MolecularApproachAuthor(s): Kelley J. Tilmon, Bryan N. Danforth, William H. Day, and Michael P. HoffmannSource: Annals of the Entomological Society of America, 93(3):640-647. 2000.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/0013-8746(2000)093[0640:DPSCIA]2.0.CO;2URL: http://www.bioone.org/doi/full/10.1603/0013-8746%282000%29093%5B0640%3ADPSCIA%5D2.0.CO%3B2

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GENETICS

Determining Parasitoid Species Composition in a Host Population:A Molecular Approach

KELLEY J. TILMON, BRYAN N. DANFORTH, WILLIAM H. DAY,1 AND MICHAEL P. HOFFMANN

Department of Entomology, Cornell University, Ithaca, NY 14853

Ann. Entomol. Soc. Am. 93(3): 640Ð647 (2000)

ABSTRACT Larvae of closely related parasitoid taxa often lack morphological differences that canbe used for species level identiÞcation. Determining the parasitoid species present in a hostpopulation may require rearing, often a time-consuming process. To monitor Þeld parasitism ratesby several species of Peristenus wasps (Hymenoptera: Braconidae) that are natural enemies of Lygus(Heteroptera: Miridae), we have developed a two-step molecular approach. Polymerase chainreaction (PCR) of the COI gene with wasp-targeting primers is performed on DNA extracted froma Lygus nymph and the parasitoid larva (if any) therein. A positive reaction indicates parasitoidpresence. A restriction digest of the PCR product then indicates which parasitoid species is presentamong known alternatives, and a diagnosis is achieved in days rather than weeks or months.

KEY WORDS Lygus lineolaris, Peristenus, parasitoid, DNA identiÞcation technique, PCR-RFLP

DETERMINING THE IDENTITY of parasitoids attacking ahost species in different habitats and locations is rel-evant to understanding both ecological and evolution-ary relationships betweenhosts andparasitoids, and toassessing biological control of pestiferous hosts. Thehost range of a parasitoid species is similarly impor-tant. Predictions about thehost range and impact of anintroduced parasitoid in a new region, with new po-tential hosts, are difÞcult to make with accuracy.Postintroduction data for already-established intro-duced parasitoids could prove valuable for developingcriteria for release programs, and shed light on naturalprocesses of geographic dispersion and host rangeexpansion.

The parasitoidÐhost associations between LygusHahn (Heteroptera: Miridae: Mirini) and PeristenusFoerster (Hymenoptera: Braconidae: Euphorinae)are good models for studying the pattern and impactof parasitoid introduction events. In North America,there are both native and introduced Peristenus spe-cieswithoverlappinggeographic andhost ranges.Thissystem can provide important data on the abundanceand impact of a newparasitoid on target andnontargethosts, and on the interaction between native and in-troduced natural enemies of a common host (Kuhl-mann et al. 1998).

Because theperiodof interactionbetween the adultparasitoid and its individual target is quite brief, Þeldstudies must often focus on already-parasitized hostsand the immature parasitoids within them. However,a frequent obstacle to obtaining parasitoidÐhost datais the issue of identity. The reduced morphology ofparasitic larvae such as Peristenus immatures can pre-

clude species and even generic determination (LoanandShaw1987).Acareful studyof larvalhead scleritesby Carignan et al. (1995) failed to reveal charactersthat separate three geographically overlappingnymphal parasitoids of L. lineolaris (Palisot), P. digo-neutis Loan, P. pallipes (Curtis), and P. pseudopallipesLoan. Also, new Peristenus species such as P. conradiMarsh (Day et al. 1992) and P. stygicus Loan continueto be introduced from abroad. Larvalmorphology alsofails to distinguish Peristenus from Leiophron Nees,another euphorine genus parasitic on Lygus and othermirids (Loan and Shaw1987,Day and Saunders 1990),further complicating the study of this system.

Studies of parasitoid populations in Lygus have re-lied on a combination of dissection and rearing (Day1994). The more easily identiÞed wasp adults areephemeral and difÞcult to sample. Dissection of aLygus subsample gives the best estimate of percentparasitism, and rearing of a paired subsample gives theidentity of the parasitoid species present (Day 1994).However, a postemergence diapause of up to 11 mo,depending on the species, precedes adult emergence.With species or generations that have long diapause,the wasp cocoons must be held in summer and thenwinter conditions for severalmonths.With parasitoidsof Lygus, it is not uncommon to wait 8 mo or more forinformation on the parasitoid species composition ina host population.

Studies on the ecological interaction of Lygus para-sitoids, as well as programs to monitor the spread ofintroduced Peristenus species, could beneÞt from asimple, rapid, and reliable laboratory assay to detectand distinguish among known parasitoid species.Though related species may be morphologically in-distinct as immatures, their molecular differences canprovide useful characters for classiÞcation. Molecular

1 BeneÞcial Insects Research Laboratory, USDA-ARS, 501 S.Chapel Street, Newark, DE 19713.

0013-8746/00/0640Ð0647$02.00/0 q 2000 Entomological Society of America

variation at the protein and DNA levels has been usedfor insect species diagnostics (Black et al. 1992; Sper-ling et al. 1995; Antolin et al. 1996; Taylor et al. 1996,1997). Furthermore, in parasitological studies, themo-lecular genetic differences between parasite and hostmay be used for parasite detection. Serological andelectrophoretic techniques have proven useful for de-tecting pathogens in insect vectors (Higgins and Azad1995) as well as parasitism in herbivorous pests(Höller and Braune 1988, Stuart and Greenstone1997). The development of the PCR technique hasfurther simpliÞed the detection of insect endobionts(Higgins and Azad 1995, Zhu and Greenstone 1999).

Thepurposeof this studywas to identify someof thegenetic variation between Lygus and Peristenus, andgenetic variation among select species of Peristenus,and use this information to develop a diagnostic toolfor the detection and identiÞcation of parasitism in L.lineolaris.

Host and Parasitoid Species. The host species in thisstudy,Lygus lineolaris, is native to andoccurs through-outNorthAmerica (Kelton1975).Oneof thebroadestherbivore generalists, it has been documented on 328plant species of 55 plant families in 30 orders (Young1986). In addition to its food plants, Lygus has beendocumented to feed opportunistically on numerouslive and dead insects (Wheeler 1976), and on theblood of faculty (Myers 1929) and graduate students(K.J.T., unpublished data).

Peristenus digoneutis, a European species parasiticon L. rugulipennis Poppius, was successfully intro-duced innorthernNew Jersey by theUSDA in the late1970s and early 1980s for biological control of L. line-olaris in alfalfa (Day et al. 1990). It has since spreadinto New York, and Þve other New England states(Fig. 1), achieving levels of parasitism well abovethose of native euphorine parasitoids of L. lineolaris(Day 1996, Day et al. 1998). Adults emerge from co-coons and are active for 10Ð14 d in the Þeld, parasit-izing early instar L. lineolaris nymphs. Usually onlyone egg is oviposited per host, and the immature para-sitoid develops over a 17-d period (Carignan et al.1995). Upon emergence from the dying mirid nymph,the larva burrows into the ground and spins a cocoon.P. digoneutis has two generations per year, some ofwhich go into overwintering diapause. Research onthe spread and impact of P. digoneutis in the northeasthas been conducted since its introduction (Day et al.1990, Day 1996). Because P. digoneutis has been thefocus of a monitoring program, it is a prime candidatefor inclusion in a diagnostic assay.

Peristenus pallipes is a parasitoid of L. lineolaris andother mirids in the northeast and other parts of thecountry as well (Day et al. 1990, Snodgrass and Fayad1991, Day 1996). Its life cycle is similar to that of P.digoneutis except that it has only one generation peryear, in the spring/early summer. Its geographic rangeincludes the rangeofP.digoneutis,making it importantto distinguish between the two. A closely allied nativespecies, P. pseudopallipes,whose single generation fol-lows that of P. pallipes, was not included in this studybecause specimenswere not available at the time. The

third Peristenus species included in this initial study isP. conradi. P. conradi prefers the introduced Adelpho-coris lineolatis (Goeze), but parasitizes L. lineolaris ata low frequency (Day et al. 1992). A fourth nativespecies parasitic on L. lineolaris in the northeast isLeiophron uniformis (Gahan); our initial efforts havefocused on the genus Peristenus and L. uniformis wasnot included in this study.

Materials and Methods

Characterization of Genetic Variability. The basisof this laboratory technique to detect and distinguishparasitoid species is that portions of the parasitoidDNA will differ from the host as well as from eachother. We extracted DNA from adult specimens of L.lineolaris, P. digoneutis, P. pallipes, and P. conradi.Wasps were collected from several locations in thenortheastern United States and authoritatively iden-tiÞed by W.H.D., and preserved at 2808C. L. lineolarisspecimens were Þeld collected from alfalfa in Tomp-kins County, NY, and preserved in 95% ethanol at2208C. We removed the abdomen from adult L. line-olaris specimens before DNA extraction to ensureagainst parasitoid contamination (though parasitoidlarvae are seldom found in adult Lygus). We followedstandard protocols for DNA extraction (Doyle andDoyle 1987, 1990) as outlined below. Specimens wereground in individual 1.5-ml Eppendorf tubes in thepresence of 23 CTAB extraction buffer and 100 mg ofproteinase K. Tubes were incubated for 2 h at 558C,homogenates were extracted with chloroform-isoamylalcohol, digested for 30 min in the presence of10 mg RNase, and then extracted again with phenol-chloroform-isoamylalcohol, and then chloroform-isoamylalcohol. The DNA was precipitated with 2.5volumes of ice cold ethanol (100%) and 1/10 volume3 M sodium acetate, washed once in 80% ethanol, andresuspended in 50 ml Tris-EDTA (pH 7.6) buffer.

We selected themitochondrial protein-coding genecytochrome oxidase I (COI) because this gene exhib-its interspeciÞc variability in other insect genera, butlow intraspeciÞc variability (Vogler et al. 1993). Themitochondrial genome has the added advantage ofhigh copy number per cell (Crozier and Crozier 1992,Simon et al. 1994).

We determined the DNA sequence for an '820 bpregion in the COI gene for L. lineolaris (Þve individ-uals), and for P. digoneutis, P. pallipes, and P. conradi(two individuals each). Initial PCR reactions wereperformed using primers C1-J-2183 and TL2-N-3014(Simon et al. 1994). PCR reactions were carried out ina Biometra Uno I machine (Biometra, Tampa, FL)using the following cycle conditions: 948C for 60 s;528C, 60 s; 728C, 90 s (335 cycles). To avoid contam-ination of PCR reactions: (1) all glassware and pipet-tors were cleaned with a dilute solution of sodiumhypochlorite on a regular basis (Prince and Andrus1992); (2) separate areas of the laboratory and sepa-rate pipettors were used for DNA extractions, DNAampliÞcations, andPCRproduct puriÞcation; (3) neg-ative controls were included in all sets of reactions.

May 2000 TILMON ET AL.: PARASITOID IDENTIFICATION TECHNIQUE 641

All PCR products destined for sequencing weregel-puriÞed overnight at 48C in low-melting point aga-rose gels (FMC, Rockland, ME). DNA was recoveredfrom '400 mg gel slices using the Promega WizardPCR Preps DNA PuriÞcation kit (Promega, Madison,WI). PCR products puriÞed this way provided con-sistently good sequence. Automated sequencing wasdone on an Applied Biosystems 377A automated se-quencing machine available through the Cornell Oli-gonucleotide Facility. All sequences were veriÞed inboth directions.

Peristenus and L. lineolaris sequences were alignedusing MegAlign in the Lasergene software package(DNAStar, Madison, WI). Apis mellifera L. sequence(Crozier and Crozier 1993) was included as a refer-ence todetermine the reading frameof the sequences.To verify that samples were not identical, we gener-ated a distance tree using theHasegawa-Kishino-Yanomodel of base substitution in PAUP* 4.0 d64 (Hase-gawa et al. 1985, Swofford et al. 1996). Based on thesealignments, we identiÞed a region that was fairly con-served among P. digoneutis, P. pallipes, and P. conradi,

Fig. 1. Range expansion of P. digoneutis in the northeastern United States, 1984Ð1996. Numbers within counties representthe year of Þrst county record. Lines represent inferred edges of distribution for 1993 and 1996. Adapted from Day et al. 1998.

642 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 93, no. 3

but highly variable between Peristenus and L. line-olaris.This region served as a basis for designing a newprimer, C1-J-2252 (after nomenclature in Simon et al.1994), [59-ATTTCYCATATAATTTWTAATGAAAG-39], which we refer to as ÔPeristenus IaÕ. This primerwas designed to selectively amplify Peristenus overLygus for this region.

Polymerase Chain Reaction of Parasitoid SpeciesUsing Peristenus Ia Primer. Using the Peristenus Iaprimer in conjunctionwith theprimerTL2-N-3014wetested the ability of this primer pair to selectivelyamplify Peristenus over L. lineolaris DNA, and theprimer sensitivity to low concentrations of PeristenusDNA. It is necessary for the primers to amplify onlyparasitoidDNAbecause they are to be used onwhole-bug extractions to indicate which L. lineolaris areparasitized. First, we tested the Peristenus Ia andTL2-N-3014 primers in a PCR reaction with P. digoneutis,P. pallipes, P. conradi, and L. lineolaris DNA, with anegative control of water in place of DNA template.Next, to test primer sensitivity, we mixed P. digoneutisDNA with L. lineolaris DNA in concentrations of 50,9, 1, 0.01, and 0.001% P. digoneutis. Aliquots of thesesolutions, and a pureL. lineolaris control, were used inPCR with the Peristenus Ia and TL2-N-3014 primersusing cycle conditions described above.

Selection and Use of Restriction Enzyme. Thealigned Peristenus sequences were used to locate sitesthat varied among parasitoid species. By comparingthe sequence of these variable regions with the rec-ognition sites of various restriction enzymes, we iden-tiÞed enzymes that would provide species-speciÞc re-striction fragment patterns when used to digestPeristenus Ia to TL2-N-3014 parasitoid PCR products.Restriction fragment-lengthpolymorphism(RFLP) inPCR products (PCR-RFLP) has been used to detectvariation within and between populations and species(Deng 1988, Chen et al. 1992, Ota et al. 1992, Aquadroet al. 1998). We chose AluI (recognition site:59..AGƒCT..39) for subsequent digests.

Peristenus Ia toTL2-N-3014PCRproducts obtainedfrom ampliÞcations of adult P. digoneutis, P. pallipes,and P. conradi were digested with AluI following stan-dard protocols (Sambrook et al. 1989) and those pro-vided by the manufacturer (Promega, Madison, WI).For each 15-ml reactionweused 0.5UofAluI and '0.2ng of PCR product. Reactions were incubated for 4 hat378Cundermineraloil andrunouton2%gels [1:1.28SeaKem LE agarose: Synergel (FMC, Rockland, ME;DiversiÞed Biotech, Boston, MA)] in 13 TBE bufferat 57v for 3 h.

Verification of PCR-Digest Identifications. To con-Þrm that the DNA protocol described above yieldsparasitoid identiÞcation results consistent with tradi-tional dissection/rearing methods, we collected L.lineolarisnymphs (28 and31 July, 1997) fromanalfalfaÞeld in Wallkill (Ulster County), NY. We randomlyassigned L. lineolaris nymphs to two groups: (1) 60 tobe analyzed using the DNA protocol, and (2) a groupto be divided anddissected (40) or reared (181) at theUSDA BeneÞcial Insect Research Laboratory in New-ark, DE, using protocols of Day (1994). Samples for

DNA analysis were stored at 2208C before extraction.We individually extracted the DNA from 60 L. line-olaris nymphs using the protocol described above.PCR was performed as described above, using thePeristenus Ia and TL2-N-3014 primers. Positive reac-tions were counted, and the DNA templates fromthose specimens were reampliÞed to provide materialfor the AluI restriction digests, which were then runon gels as described above. The resulting bandingpatterns were compared with positive controls (di-gests of identiÞed Peristenus spp.). Parasitism ratesobtained from the two methods were compared usinga comparison of binomial proportions with normaldistribution (Ott 1993).

Results

Characterization of Genetic Variability. Align-ments were unambiguous and no insertions/deletionswere observed (alignments are available from the au-thors). Sequences are deposited in GenBank (acces-sion numbers AF189240, AF189241, AF189242,AF189243). We chose a region 26 bp long as a basis forthe Peristenus Ia primer (above).

For the 820 bp region sequenced for L. lineolaris, P.digoneutis, P. pallipes, and P. conradi,we found speciessequence divergence as presented in Fig. 2. This con-Þrms that the Peristenus sequences are more similar toeach other than to either A. mellifera or L. lineolaris,and that sequences were not contaminates of eachother.

Fig. 2. Distance tree based on HKY85 substitutionmodel. The same tree topology is obtained irrespective of thesubstitution model selected. Numbers along branches indi-cate branch lengths.

May 2000 TILMON ET AL.: PARASITOID IDENTIFICATION TECHNIQUE 643

Polymerase Chain Reaction of Parasitoid SpeciesUsing Peristenus Ia Primer. The primer designed inthis study is intended for use with DNA extractions ofL. lineolarisnymphs todeterminePeristenusparasitoidpresence through positive PCR (with subsequentidentiÞcation). For such, it is important that theprimer pair (1) ampliÞes only parasitoid, and not host,DNA, and (2) detects a very low concentration ofparasitoidDNA so that the earliest stages of parasitism(e.g., parasitoid eggs) are not overlooked.

Using the Peristenus Ia primer in conjunction withTL2-N-3014 we obtained positive PCR for each Peris-tenus species, but not for L. lineolaris (Fig. 3). PCR ondescending concentration of P. digoneutis (relative toL. lineolaris) DNA was positive at 50, 9, 1, and 0.01%P. digoneutis, and the 0.001% P. digoneutis and the L.lineolaris control DNA did not amplify (Fig. 4). Wecalculated that even a newly laid parasitoid egg com-prises at least 0.07% of the host, a level well above theminimum threshold (0.01%) of primer sensitivityfound here.

Use of Restriction Enzyme. Based on the AluI rec-ognition sequence, predicted band lengths for restric-tion digest on Peristenus Ia to TL2-N-3014 PCR prod-ucts are P. digoneutis, 180, 188, and 393 bp; P. pallipes,42, 66, 80, and 574 bp; P. conradi, 42, 80, and 644 bp.When we used the restriction enzyme AluI to digestthePCRproduct fromthePeristenus Ia toTL2-N-3014reaction, observable bands on the gel (Fig. 5) rangedfrom184 to 644 bp. Predicted bands smaller than '150bp are not resolvable under these conditions. How-ever, bands .150 bp provide a species speciÞc patternthat variedamong the3Peristenus species in this study.

VerificationofPCR-Digest Identifications.Of theL.lineolaris nymphs processed using the DNA protocolsdescribed in this article, we found that 33.3% (20/60)were parasitized, all by P. digoneutis. Dissection re-

vealed a 32.5% (13/40) parasitism rate. The two par-asitism rates were not signiÞcantly different (SE 50.096; P , 0.05). Rearing revealed a 13.8% (25/181)parasitismrate: 23P. digoneutis,1hyperparasite (prob-ably Mesochorus curvulus), and 1 dead (and thereforeunidentiÞable) parasitoid larva.

Discussion

The technique developed in this study can be usedto identify and quantify the known parasitoid speciesin a host population. Although this technique wasdeveloped to detect Peristenus parasitoids of L. line-olaris, it can be expanded to include additional taxa, orcan be adapted to different parasitoidÐhost systems,giving it broad applicability. The two main steps in theprocedure are as follows: (1) DNA extraction of in-dividual potential hosts and PCR with parasitoid-tar-geting primers, and (2) restriction digest of positivePCR products. The technique reduces identiÞcationtime in theL. lineolaris-Peristenus system frommonthsto days; further reÞnement may reduce this time evenfurther. It eliminates the need to laboratory-rear sam-ples, and also allows for the storage of samples until itis convenient to analyze them.

The parasitism rates revealed by the DNA and dis-section protocols are consistent with each other, sug-gesting that the two methods are equally accurate.Rearing and identiÞcation of adult parasitoids con-Þrmed that the species identiÞcations obtained usingthe DNA protocols were also accurate. The discrep-ancy between parasitism rates revealed by rearing(13.8%) andDNAor dissection (33.3 and 32.5%) is notunexpected, because a higher mortality of parasitizedindividuals is not unusual in the rearing process [a factthat has previously necessitated paired dissections(Day 1994)].

Fig. 3. Bands produced by PCR using Peristenus Ia andTL2-N-3014 primers.

Fig. 4. Bands produced by PCR with Peristenus Ia andTL2-N-3014 primers on varying concentrations of P. digo-neutis DNA relative to L. lineolaris DNA.

644 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 93, no. 3

Apotential pitfall of this technique is that restrictionsite variation could lead to false readings for new orunexpected parasitoid species or conspeciÞc variants(Sperling and Hickey 1995, Sperling et al. 1996). Fur-thermore, primer mismatch for new or variable spe-cies could prevent the initial ampliÞcation that indi-cates parasitoid presence. To reduce the possibilitythatnew,unexpected, or variable species couldescapedetection through nonspeciÞcity in the restriction di-gest, it would be good practice to routinely select arandom subsample of PCR ÔpositivesÕ for direct se-quencing to verify identity and screen for variation.The danger of encountering the latter problem ofprimermismatchcanbe reducedbyusingprimerpairs

effective at the family level for both braconids andichneumonids. The Peristenus Ia and TL2-N-3014primer combination does in fact amplify a variety ofHymenoptera including bees, the 7 Peristenus speciestested thus far, other braconids, and ichneumonidsincluding M. curvulus (a hyperparasite that will ulti-mately be incorporated into the assay). However, theprimers do not work universally within the Ichneu-monoidea.Weplan todesign anewprimer speciÞcallyfor the Ichneumonoidea to replace TL2-N-3014,which could help ensure universal ampliÞcation ofhymenopterous parasitoids. The presence of dipter-ous parasitoids, if suspected, would be revealed bestthroughdissection; however, nonehavebeen found inmirid nymphs to date (Day 1995).

The Lygus lineolaris–Peristenus digoneutis system isuseful for careful study because its successful intro-duction to the United States 15 yr ago can help answerquestions about what has happened in the years afterthe introduction. The ease of detecting particularparasitoid species makes the technique outlined hereuseful for monitoring geographic range expansion ofan introduced parasitoid like P. digoneutis, and forassessing parasitoid activity in previously unstudiedhabitats or crops. It can aid in assessing parasitoidimpact on target andnontarget hosts and also in study-ing the interactions of native and introduced parasi-toid species. The technique is particularly useful insituations where multiple hosts are under study (aswith assessing impact on nontargets), because theeffort required to rear different hosts and parasitoidsmust certainly limit the array of different host speciesand samples which can be maintained in the lab; in-stead, potential hosts and other samples can be col-lected, identiÞed, and stored for laboratory analysis.

Acknowledgments

We thank S. Ji for advice and assistance, T. Wood, F.Sperling, and an anonymous reviewer for helpful critiques,and D. Swofford for providing a test version of Paup* 4.0 toB.N.D. This project was supported in part by National Sci-ence Foundation research grant DFB-9508647 to B.N.D., andgrants to K.J.T. and M.P.H. from USDA-ARS (Cooperativeagreement 58-1926-5-042), and from the New York Stateintegrated pest management (IPM) Program, the NorthAmerican Strawberry Growers Association, and the NewYork Berry Growers Association.

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Received forpublication33March1999; accepted19October1999.

May 2000 TILMON ET AL.: PARASITOID IDENTIFICATION TECHNIQUE 647