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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:
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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
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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
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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.
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