V O L .28 N O .1
S O U T H W E S T E R N E N T O M O L O G IST
M A R .2003
ARTIFICIAL FEEDING SYSTEM FOR TI{E SQUASHBUG,
ANASA rflSz.t @E GEER) (I{ETEROPTERA:COREIDAE)
B.D. Bruton2,S.D.Pail,
Blake R. Bqrtiner , Astri Wayadandet,
ForrestMtchell3, andJacquelineFletcherr
ABSTRACT
Squashbugs, Anasa /rislis (De Geer) (Heteroptera:Coreidae),did not feed on
or on pouchliquid diet-filled feedingsachetsusedtraditionallyfor feedinghomopterans,
like artificial feedingsource(AFS) containinga meridic diet developedfor rearingthe
western tarnished plant bug, Lygus hesperusKnight, another heteropteran. However,
ofthe desireddiet and
excisedcubesofsquashfruit, vacuuminfiltratedwith a suspension
offeredabovea screenbarrier,wereaocepted.During a 48-hrtestingperiod,all cube-fed
adult insectssurvived,while 35% of thosefed on the meridic diet offered in parafilm
pouches and 40o/oof those fed on 5% sucroseoffered in sachetsdied. After blue food
coloringwas addedto the dietsas a marker,the excretoryfluids of 75% of insectsfed on
infiltrated cubeswas blue in color indicatingthat feedinghad occurred,while no blue
defecationoccurredwhen dye-amendeddiets were offered in the other two systems. The
A. tistis AFS provides a convenientsystemfor studyingthe feeding behavior ofsquash
bugs and provides an alternativeto whole plants for studying the etiology of cucurbit
yellow vine disease.
INTRODUCTION
The squash bug, Anasa rrisfis (De Geer) (Heteroptera: Coreidae), occurs
throughoutNorth Americaand is considereda majorpestof cucurbits.A. tristisfeeding
involvespiercing ofthe plant's epidermisby the styletsand intracellularpenetrationto
'lacerateand flush'
the mesophyllor vasculartissues@eard1940,Bonjour l99l). This
method of feeding intemrpts xylem transportof water due to vasculardamageand
blockagethat resultsin collapseof plantstructuresdistalto feedingsites(Neal 1993).
Cucurbit yellow vine disease(CYVD) is characterizedby rapid and general
yellowing of leavesappearingovera 3-4 dayperiod,followedby gradualor rapid decline
and death of the vine in severalcucurbit crops (Bruton et al. 1998). A. tristis was
reported(Pair et al. 2000)andconfirmed@extineet al., unpublisheddata)to be a vector
of Serratia malcescens,the causalagent of CYVD (Bruton et al., in press). This
bacteriumhasbeenculturedfrom diseasedfield-grownwatermelon,zucchini,pumpkin,
andcantaloupe.
l
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P resent address of senio r authort D ep artm ent of E nto m olo g y, U niv of C alifornia,
R iversid e , C A 92 507
Although artificial
-feeding systems(AFS) have been developed for many
homopteraninsects including
leafhoppers,aphids, and whiteflies, toiw have been
developedfor heteropterans
(cohen 2oo0b). AFSs,alongwith artificial diets,havbbeen
developedfor the westerntarnishedplanf bug, Lygus iesperus Knight
rconen .2000a;
Debolt 1982; Debolt and patana 1985), thJ reduviid big, Triatoina in/estans
Klug
(schaub l99l), and the southemgreen stink bug, Nezara iiriauu (L.) (Ragsdale
* al.
1979). In the latter case,the AFS was instrumentalin associating'insect
rlahg wittr
microorganismtransmission.
The relationship betweenthe vector A. tristis and the pathogenS. marcescensis
.
an important elementof the etiology of cyvD, but investigationiof pathogen-vector
interactions.were hamperedby the lack of a convenient-AFs uy wtrictr-pathogen
acquisitionby the squashbug could be assuredand controlted. preliminaryanemptsto
feed l. tristis on known homopteranor heteropteran
artificial diets were unsuccessful.
The goal of this studywas to developan AFS and a suitablediet on which to maintain
squashbugs during researchstudiesand to providea convenientacquisitionsourcefor
the microorganism.Feedingacceptance
andsurvivorshipof A. rzisrison traditionalAFSs
were comparedwith thoseon a new A,FScomposedof diet-infiltrated squashcubes.
MATERIALS ANDMETHODS
colonies of A. rristis,initiatedwith adultscollectedfrom a field nearLane, oK,
were maintainedin screenedcages(50 x 25 x 45 cm) in a growth room at 27"c,
12L:12D, and 45-50%o
RH. colony insectswere rearedon purptin plants(Cucurbin
pepo L. var. pepo "connecticut Field") that were replacedat- weekly intervals and
supplemented
with washedzucchinisquashfnrit (C.pepoL. var.melopefo).
SeveralAFSsandartificial dietsusedfor-sustaining
otherhemiptlraninsectswere
offered to A. trislis and observationsof feeding were made. Feeding sachets,or
stretched-parafilm
membranefeedingsystems,commonlyusedfor studyi;g leafhopper,
aphid,and whitefly feeding(Mitsuhashiand Koyama l97l), hereafterrerenedto as the
lromopteranfeeding system, consistedof 29.5m1flexi-cup medicine cups (Baxter
Heathcarecorp., Deerfield,IL) coveredby one layer of parafilm (AmericanNational
can, Greenwich,cr) stretchedto 4X its original sizeover the openingof the medicine
cup. A volume of 0.5m1of 5olosucrose(pH 7.0) was placedon the outer surfaceof the
stretchedparafilm and coveredwith anotherlayer of stretchedparafilm to enclosethe
solution (Mitsuhashiand Koyama l97l). Twenty insects,not separatedby sex, were
placedinto the AFS, one per sachet,andthe sachetswere maintained at 27oi, l2L:12D,
and45'5o%oRH. Anotherfive insectswere individually offereda modificationof this
membranefeedingsystemin which squashslurry,madeby lique$ing approximatelyl0g
squashfruit and 20ml steriletap water in a blender,was brushedonto the insect-facing
surfaceof the membrane(lml squashslurry per l6cm2membranesurface)as a possibli
gustatorycue.
A non-membrane
systemwas also iestedby offering semi-solidpreparationsof
5%oagaroseor 3ologelatin. Ten ml of solutionwas pouredinto the bottomof eachol'five,
60 x l5 mm petri dishes@agesdaleet al. l97g). A 70x20mm, fiberglassscreenbarrier
with 1-mm grid squareswas placedso that it restedon the rim of the petri dish. Five
insects(sex not determined)were placedon top ofthe screenin eachdish, and the dish
lids were positionedon top, their rims restingon the screen.The disheswere maintained
for 48 hr at27oC,constantlight, and45-50%RH.
Intact squashfruit were washedwith handsoapandreverseosmosis(RO) wateq
and subsequently
was cut with a sterilerazorblade. The epidermiswas removedand 6mm' cubeswere excisedfrom the cortex. Squashcubeswere vacuuminfiltrated with
blue dye so evidenceoffeeding could be determinedby evidenceofblue dye in squash
御
bug excretory fluid. For infiltration, squash cubes were submerged in RO water
coitaining blue food coloring(McCormicli and Co., Inc, Hunt Valley,,MD) (O'lml per
tOml HzO) at a rate of I cubeper ml in a 500-mlErlenmeyervacuumflask.. A vacuum
force was applied for 5 sec, during which negativepressureforced air from the
intercellular ipaces of the tissue, and as the vacuum was quickly releasedthe bluecoloredwater enteredthe intercellularspaces.
Adult squashbugs were confined individually with a squashcube using an
apparatussimilar to that?escribedby Ragsdaleet d. (1979). The insect-wasplacedin
th" botto. half of a 60 x 15 mm petri dish. A 70 x 70 mm squareof sterile l-mm
fiberglass screenwas placedover the dis[ the squashcubewas positionedin the center
ofthl screeq andthe petri dish lid was positionedon top. The squashcubewas offered
abovethe insect,ratherthan below, to avoid excretoryfluid contaminationofthe cube'
The feeding apparatuseswere incubatedin a humidity chamber(placedon a stand in a
coveredtransparentplastic shoeboxcontainingwater at a depthofapproximately lcm) to
minimizecubedesiccationandwereheldunderconstantlight x27"C.
Threeof the AISs (the homopteranmembranefeedingsysteq the tarnishedplant
bug feeding system, and ihe newly developedinfiltrated squashcube 4pS described
ab6ve; amendedwith identical ratios of blue food coloring were tested for A, tristis
acceptanceover a 48 hr period at 27oC,constantlight' and45-50ploRH.
In the homopteran membranefeeding system, 20 adult A. ttistis were placed
and given accessto 0.5m1of 5olosucrose(pH 7 0)
individually into the apparatuses
containing'bluefood coioring(0.lml per loml sucrose)(MitsuhashiandKoyamal97l).
Tf,e tarnishedplant bug feedingsystem(Cohen2000a)consistedofmeridic diet,
a lima bean meal and wheai germ basedformulationcontainingadditional nutrient
andcontainingblue food coloring(O.lml per l0ml diet) enclosedin l00mm
supplements
* Zir-. unstretchedparafilmpouchesas describedby Debolt and Patana(1985). This
by sex),with the parafilm
diet was offeredindividuallyto 20 singleinseots(not separated
plateswith 35-mmwells'
-pouchessupportedon meshscreeningin six-welltissueculture
The A. tristis feedingsystemwas also offeredindividually to 20 A. '/t'rtis (not
by sex). For all threi feedingsystemsfilter paper(Whatmanno. 4), cut to fit
separated
the bottom of the apparatus,allowed collection of the droplets of squashbug excretory
fluid. If this fluid was blue, squashbugswere assumedto havefed on the offered diet.
Insectmortalityoverthe 48-hrperiodwasrecorded.
for boih detecrionofblue excretoryfluid and insectmortality, chi squareanalysis
differences(criticalP-value=0.05)betweentreatments(SAS 1996).
wasusedto assess
RESI.JLTSA}ID DISCUSSION
In our preliminaryexperiments,when squashbugswere placedin severalAISs
and offered artificial diets known to be acceptableto other homopteranand heteroperan
insects,neitherstylet insertionnor test probingwas observed.Theseinsectsdid not feed
throughany ofthe testedmembranebasedfeedingsystems,nor would they feed on the
semi-solidagaroseor gelatinformulations.
Squashbugs ied on blue, water-infiltratedsquashcubeswithin one hour of
introduction,with 15 of 20 test insectsexcretingdropletsof blue fluid onto the filter
paper after 48 hr (Table l), whereas none of the insects offered the other two
dF-'A,P<0.001). None of the twenty
systemVdietsexcretedany fluid (chi-square=40,
AFS died during the 48-hr period,
in
cubes
our
water-infiltrated
blue
insects offered
comparedto 7l2Oof those offered the meridic diet in unstretchedparafilm bags and 8/20
were significantly
of those offered 57o sucrosein feedingsachets. Thesepercentages
different amongtreatments(chi-square=I 0. I 3, dF2, P<0.0 I ).
６
鮒熱撚齢 襴 襴 鰍 i路麒ぢ i鮮
TA B LE l
th e
Preferenceandsuitability of ThreeDifferentArtificial Feeding
Systemsby
Bug,Anan tristis.
Treatment"
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亀
Sy Stem w ith M erid ic
卯
artiflcial d iete
F eed in g Sach ets w ith 5%
B lu e E x cetory F lu id a
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N ot B lu e
15
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D ied
20
0
0
20
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testingS.
be expectedin the plant. Becausethe CYVD pathogenis phloem-associated,
^*"it"r^
transmiisionby A. tristisin the artificially inoculatedsquashcubeAFS may
not exactly mimic acquisition or inoculationof the pathogenin a naturally infected plant
system. The vacuuminfiltration of a squashcubesaturatesall intercellular spaces,.which
would not occur in plant infection, and doesnot assurebacterialentry into phloem sieve
tubes. However, ttie AFS developedfor A. tristis providesa tool that will facilitate the
studyof CYVD.
ACKNOWLEDGEMENTS
RegionalIPM Programandthe
This researchwas fundedby the USDA-Southern
Oklahoma Agricultural Experiment Station. Thank you to Edmond Bonjour and
KristopherGles for reviewingthis mamrscript.
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595-682.
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Parameters
S8,
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