Archivesof InsectBiochemistry and physiorogy 21 2271-280 (tggz)

Roleof chillingin the Acquisitionof cold Toleranceand the capacitationto Express stressProteins in Diapausingpharate Larvae of the CypsyMoth, Lymantriadispar

David L. Denlinger, Richard E. Lee, Jr., George D. yocum, and Olga Kukal Dcpartmentof Entomolagy,ohio statelJniuersity, columbus (D.L.D., G.D.y., o.K.), and Departmentof Zoology,Miami unioersity,Oxfoid (R.E.L.), Ohio

Cold hardinessin eggs(pharate first instarlarvae) of the gypsymoth is not a componentof thediapause program, but is acquiredonly afterthe pharatelarvae havebeen chilled. The supercooling points of unchilled(2yC)and chilled (5.C) e88sare nearlythe same(ca. -27'C\, and chillingdoes not furtherelevate concentrationsof glycerol,the majorcryoprotectant, yet chillingat 5.C greatly increasesthe pharatelarva's tolerance of -2trC. One conspicuousdifference betweenthe chilledand unchilledpharate larvae is theirability to expressstress proteins. The most abundantlyexpressed stress protein, 25,000 Mr, was ex- pressed morehighly in chilledpharate larvae than in unchilledpharate larvae. bothat hightemperatures () 40"C)and in responseto low temperature(-r 5"c). Thiscorrelation suggests a link betweenstress protein synthesis and the acquisition of cold tolerance. .o 1992!\,itev_Liss, Inc.

Key words: cold hardiness,diapause, heat shock proteins, supercoolingpoint, glycerol

INTRODUCTION

The gypsymoth, Lymantrindispar, is amongthe relativelyfew insectswith an.obligatorydiapause. Though a few first iristarlarvae ao hatchfrom eggs in latesummer without the interventionof diapause [7,2],most halt devei6i- ment aspharate first instarsand do not hatchuntil the foilowingspring. This diapauseis geneticallyprogrammed and the developmentaldecision to enter diapausedoes not appearto be influencedby photbperiod,temperature, or other factorsthat commonly serveas environmentai regulators'ofJlufu"r"

Acknowledgments:This work was supp_ortedby U.5. ForestService cooperatrve agreement 23-0gg from NortheasternForest ExperimentalStation,'uSDA-CRCO Cruni si-:z:0-6223, and NSFCrant DCB-881r 317.

ReceivedJune B, 1992;accepted Seprember B. 1992.

Addressreprint requeststo,David L. Denlinger,Departnent of Entomologv.ohio St.rteU'i'ersrty, 1735 Ncrl Ar tnue Colunrltus,OH -r;: tO

. lqq ) I,t iit,.._[ is.. ipr _ 272 Denlingeret al. in its induction [1]. Environmental conditions, however, do play a criticalrole termination. A period of chilling is essentialfor diapausetermination in this species^ [3,4]. Gypsy pharate first instars, though intolerant of freezing, are weil uaaitea to survive the low temperaturesof winter [5-7], but it is not clear whether chilling is required to elicit cold hardiness.Is a diapausingpharate already .o'id hut,iy, or is this attribute only acquiredin responseto low in the literature temperature-In exposure? Examples of both possibilitiesexist development t8]. this study, we examinethe importanceof chilling in the of cold hardinessby comparing supercoolingpoints, cryoprotectantlevels, and toleranceto low temperaturein unchilled and chilled samples.The recent discoverythat diapausingpharate first instarsof the gyPsymoth canrespond to both high ur,d lo* temperature extremes by synthesizing heat shock proteins [9] suggeststhat these stressproteins may alsocontribute to over- wintering success,and in this study we demonstratethat chilling has a role in capacitatingthe gypsy moth pharatelarvae to synthesizestress proteins in responseto temperaturestress.

MATERIALSAND METHODS

Insects Newly deposited gypsy moth egg masseswere obtained from the Center of BiologicalControl, Forest Service, USDA, Hamden,CT, and storedin Petri dishesitzS'Cfor at leastone month to permit the completionof embryogene- sis.When the diapausestage (pharate first instarlarva) was attained,the egg masseswere either held at 25"C (unchilled)or 5'C (chilled).

Measurementof Cold Tolerance Groupsof 25 pharatelarvae were placedin individual testtubes immersed in a -20'C ethanoi bath within a freezer,and test tubes were removed at variousintervals. Samples that had previouslybeen chilled at 5'C for 100days were transferreddirectly from -20 to 25'C (15h light:9h dark cycle),but those that werepreviously unchilled were transferredto 5"C for 100days of chilling beforebeing placedat 25'C. Hatching successwas recordedfor eachgroup.

SupercoolingPoint Determination SCPs*of individual pharate larvae were determinedby positioning a 30 gaugecopper-constantan thermocouple in contactwith the chorion.A cooling iate of ci.-1"C/r.,inwas maintainedusing a low temperafurebath. The SCP was the lowest temperaturerecorded prior to the releaseof the latentheat of fusion.

.,\bltreviatrons used: SCP = supercooling point: NNIR = nucle.lr rlr.rqnettcresorrance; 5D5 = sodiunrdociecr | \ult.tl(. GypsyMoth Cold Tolerance 273 TLC Separation Pharate larvae.from a single egg mass(N : ca. g00eggs) were ground with mortar-and pestleand.centrifrlqed at 1,0009for 1 min]ihu ,upu"rnatantwas spotted on precoatedsilica gel F25aplates 0.25 mm thick (Mer&, Darmstadt, Cermany)using fwo different solventsystems: solvent A, butanol:acetone: water' 4:5:1;solvent B, isopropanol:ethyl acetate:water, g3:11:6.The plates were visualizedby. ultraviolet light and also by spraying with 1:1 sulfuric acid:water.The following standirds were run *itn .u.fianalysis: alanine, glutamine,proline, ethyleneglycoi, grycerol,sorbitol, and trehilose. HPLCSeparation

Concentrationsof glycerolwere determinedas describedby Lee et al. using [10J HPLC (WatersAssociates, Milford, MA). For eachdetermination, 5lgg masseswere homogenizedin 3 ml ethanoland centrifuged3 timesat 2,00b! for 5 min each.Each time, the pellet was reextractedwith 3 ml methanoland pooled supernatantswere_passed thro,gh a sep-pak Cre cartriJj* Associates) fwut"r, and evaporated.to dryness.simplur^*ur" resuspend"i i" 0.5 ml ethanol:water(1:1), filtered through a 0.22 pm nylon filte; (MSI, Honeove Falls, NY) and injected into a taaiaily .o*prurred silica .bt.r..rr.iw",*, Associates)modified with tetraethylenipentamine. NMR Analysis lH-Decouplgq 13c . NMR spectra were obtained on a Bruker AM-500 Fou- rier-transform NMR specrrometerequipped with a pulse progru.rr^er and quadrature phasedetection. Spectra',uere-generated with Z0pu6e widths and a-recycletime (acquisition* relaxationdelay) of 1 s; 16*32Kreal points were obtainedover a o t90 lg ppm speclralwidth. Chemicalshifts u."'r.poited in (partsper miliion)relative to the PP,S''C Cl signaloi deuterochloroforrn. . Nr\lRsPectra oi intaci eggs(pharate larva and undigestedyoik encased within the chorion)were determinedby insertingca. 20 elgs int6 a s mm i.d. NMR tube.Spectra of homogenateswere obtainel by anallizingthe superna_ tant collectedfrom a homogenateof ca.g00 eggs, us descritua]uouu.ill-iic spectrawere comPared to standar-dchemical -glvcol shiftsdetermined previouslv [11] and to the standardfor ethvlene (63.3ppm). ProteinLabeling and Separation Pharate larvaewere separatedunder salinefrom the restof the eggby using a.forceps to mechanicailystrip off the chorion and remainingyoil"1ti1. rirre pharatelarvae were bisectedind cultured togetherin a t.S mlviat containlng 10 pl methionine-free Grace's culture meJitrm [13]. curtures to be rreat shockedwere placedin a water bath-atthe heat shocktemperature and then labeledusing 1 pci d (iq [370.k!ll)35s-t"ueledmethto"i"e iriur.,, *s_tiur,,,, sqe.9i{icactivity 7,259Cilmmol, ICN Radiochemicals,Costa Meru, c{io, u. additional t h at the same temperature.Cultures to be cold shockei were exposedto -15"C in a.low temperaturebath ior24 h, hansferredto 4'C for variousintervals, and labeledas abovefor an additional24h at 4"C. The Crace's meclium was then removeci. the tissue homogenizecl r,vitha c-lispos.rbie 274 Denlingeret al. pestle (Kontes, Vineland, NJ) in 20 pl sarnplebuffer (0.67oTris, 10% P- mercaptoethanol,br_omophenol blue), boiled for 5 min, and storedat -70"C. The amount of 35Sincorporation for each samPle was determined by trichloroacetic acid precipitation [14]. SDS-PAGE was run using equal amounts of radioactivityper sampleon a discontinuous10% gel [15]with a modified stackinggel [16].Gels were run at 15mA, fixed, and stainedusing a modified Coomassieblue method [17], destainedwith aceticacid:metha- nol:water(0.18:1:1), and saturatedwith 1 M sodiumsalicylate [18]. Cels were dried onto Whatman filter paper and exposedto Kodak X-Omat X-ray film (Rochester,NY) at --70'C.

RESUTIS

Low TemperatureSurvival To determine whether chilling enhanceslow temperaturesurvival, unchilled pharatelarvae and thosethat had beenchilled at 5"C for 100days were exposed to -20'C challengesof variouslengths (Fig. 1). The unchilledpharate larvae were held at 2fC for one month beforeexposure to -20'C and then chilled for 100 daysat 5'C beforebeing retumed to 25"C.In contrast,the chilledpharate larvae were held at 25"C for one month, chilled at 5'C for 100days, then challenged with -20'C, and thereafterplaced at 25"C. Chilled pharatelarvae were clearly more tolerantto -20"C than pharatelarvae that had not beenchilled.

SupercoolingPoints The SCPsof newlv depositedunchilled diapausing pharate larvae held at 25'C were aiready \ow (-27.7"C),and chilling at 5"C did not alter the SCP (Table 1). Though the SCP of the sample chilled at 5'C for 55 days was significantlvdifferent from the others, the differencewas siight and is not

100

380 o I o E60 o 6 I rn )6'v ;g-20

Tim€ at -2ooC (days)

Fig. 1. Successof larvalemergence (mean t 5.E.of 3 groupsof 25 eggs)from eggsthat were exposedto -20"C for various numbers of days. The egg masseswere first held at 25"C for one month to permitcompletion of embrvogenesis.One grt>upthen receivedthe -20'C exposurebefore beingheid at 5'C for 100 days(open circles), and the c.rthergroup was chilledat 5"C for 100 days and then exposedto -20"C (solidcirclest. All sampleswere then transferredto 2-5'Cto monitor hatch ing Cypsy Moth Cold Tolerance 275

IABIE 1. Comparisono-f Supercooling Points and GlycerolConcentrations in L. disparin One-Month'Old Unchilled DiapausingEggs and Eggi That WereHeld Unchilled for One Month and SubsequentlyChilled at 5'C for Variouslntervalsr

scP('C) Glycerol(mM) Chilling status mean + s.E. n meant S.E.

cl -27.7 Unchilled, 25'C * 0.1a 3 9.9+ 7.4a -26.9 Chilled, 5"C for 55 days 15 * 0.2b 3 3.9+ 1.1b Chilled, 5'C for 100days 10 -25.5 t 1.0a Chilled, 5"C for 55 days, then to 25'C for 7 davs i1 -27.0 '+ 0.7a tUnchilled = diapausing eggs pharate larvae and yolk encasedwithin the chorion. Means within each column followed by the same letter are not iignificantly different; ANOVA, ScheffeF-test for SCP comparisons; t-test for glycerol comparisoi, p < 0.6S.

likely to be biologicallyimportant. Likewise, the SCP was not altered by transferringPharate larvae that had beenchilled at 5'C for 55days to 25"C for 7 days.

Cryoprotectants

__l*o potential cryoProtectants,glycerol and trehalose,were detectedby fLC gnllysisof eggs(pharate larvae and yolk) that had beenchilled at 5.C for 100days. "c NMR spe-ctraof intact, chilled eggs(Fig. 2A) indicatedthe presenceof saturated and unsaturated lipids (reJonanlesat 30, 130,and 175ppm) and-phospholipids (ss-7s ppm), and the presenceof glycerol (resonancesof 65and 75 pprn),treharose (62,7r,73, i4, and 95ppirj, ana glucose(10 peaksof ranging between 62.2 "- ild ?pll",lg:er3c and'ci7.4ppm) could be verified bv the NMR speitrJ of the supernatantoi egg homogenates(Fig. 28). Glycerol was the only- potential cryoprotectantidentified by HpLC in unchilledeggs and.eggs that had beenchiilea at 5'C for 55 days.toncentra- tions in both unchilled and chilled eggs were low (Table1), and there is no evidencethat glycerol concentrationsincrease in responseio chilling. StressProteins

The capacityof chilled and unchitled pharate larvaeto synthesizestress proteins was evaluated by comparing the responsesof these two types of pharatelarvae to heatshock and low temperatuie.At high temperaturiJ,httle differencebetween.the two ty?es of pharatelarvae was apparentat temper- aturesbelow 40"c (data not shown), but at both 4L and ,igic, the 75,00dM. protein,a protein immunologicallyrelated to the human 70,000M.heatshock protein [9J,was more strongly expressedin the chilled pharateiarvae (Fig. 3A, comparelanes 2 and 4 with lanes1 and 3 from unchilledpharate larvae). Amo.ng the 7 geis of.th.tr ryp:-that were run, the chilled pharate larvae consistentlyexpressed the 7s,000M. protein more highly ihan unchilled pharatelarvae at high temperature,and in all casesthe-diiferences were at leastas pronouncedor much more pronouncedthan in the example,ho*. in Fisure34,. 276 Denlingeret al.

r80 150 140 120 100 80 60 40 20 PPX

r80 r60 1,{0 120 100 60 a0 PPX

'3C Fig. 2. NMR spectraof (A) intacteggs (i.e., diapausingpharate larvae and yolk enclosed withinthe chorion) and (B) centrifugedegg homogenates of thegypsv moth. The diapausing sampres were chilled at 5"C for 2 months and spectrawere recordedat 25'C. Spectraof intacteggs show prominentbroad resonances at ca. 30, 130, and 175 ppm which correspondto lipids,and j5-70 ppm correspondingto phospholipids.Spectra of homogenatesindicate the presenceof trehalose representedby 6 carbon resonancesbetween 62-95 ppm, glucose(10 peaksof a- and b-pyranose rangingbetween 97.4 and 62.2) and glycerol (65 and 75 ppm). Peaksarising from resonances around the 30 and 130 ppm regionsshow contaminationof the centrifugedsample with saturated and unsaturatedlioids. resDectivelv.

Likewise, pharate larvae that had been chilled at 5'C for 65 days were more resPonsive to a -15"C exposure than unchiiled pharate larvae. After a 24 h exposure to -15'C, chilled pharate larvae expressed the 75,000 M, stress protein to a greater extent than unchilled pharate larvae (Fig. 3B). This differencewas apparent within 48 h after -15"C exposure(compare lanes 1 and 2), anclthe differencepersisted forat least14,1 h (cornparelanes 5 and 6). Cypsy Moth Cold Tolerance 277 A

-75

-75

Fig.3- stressprotein expressionby diapausingpharate rarvaern responseto (A) erevated temperaturesor (B)during recovery from cold shock.Pharate larvae were either unchilled (35 days at 25'C) (A: lanes1,3; B: lanes1,3,9,7,9) or chiiled at -1'C for 65 days(A: ranes2,4; g: ranes 2,4,6,8,10)before exposure to theerperimental temperatures. For the hieh temperature erperrrnents (Ar,pharate larvae lvere dissectedrrom the chorronsancl exposed to +l (lanes1,2) or -13"C(lanes 3,4) for I h, then labeled for I additionalhour at the sameiemperature. For the low temperature experiments(B), chorionated pharate larvaewere exposedto-is"C ior 24 h and then afier.lg h (lanes1'2),96 h (lanes3,4) (lanes or 144 h 5,6) at 4'C, the pharatelarvae were dissected from theirchorions and labeled 24 h at 4'C. Controlswere labeledat.d0"C (lanes 7,g) and 25"C (lanes were separated jj::1ns on r 0% sDS-pAcE gers M, of the malor srressprorein is expressed In:: l?l KilOOattons.

DISCUSSION

In some , including the silkmo th Bonfuyxmori [lgl, Hyalophoracecropia [20],and the flesh sarcophngacra,ssiparpis lzTl-, cold hardin"r', i, iir*ty ii.,t.a to the expressionof diapause.The two events cannot be separaiedand apparently sharea commongenetic basis for inducdon.This ao"" .,ot appear to be true for the gypsy moth. our experimentsdemonstrated that cold hardiness, measuredas toleranceto -20;c, is not inherent to the Jiupu,rru program. Rather,it is acquiredin responseto chilling.This impliesthat'eggs, laid.during the summer,lack cold hardinessat firit and onlv becomecold haldy in response to the of autumn.Though aiupu,.,s" o,.,a colc-l low femperatures hardinessclearlv coincide cllring the rvintermonths, these trvo events 278 Denlingeret.al. do not sharea common inductive Pathway.This, of course,does not mean that the diapauseprogram is not involved in preparing-the-Pharatelarva to respond to ittitting, and this is indeed a iikely-scenario[8], but frgm this set of experimentsit L clear that the induction of diapause,by itself, does not .u.,r. the gypsy moth to becomecold hardy. in this respect,the gypsy moth is similar t" thd parasitoid Braconcephi 1221, the Europeancorn borer Ostrinia nubilalis[23], and the Vicerovbutterfly Limenitisarchippus [zal: The mechanism(s)used by gypsy moth eggsto achievecold hardinessis still unclear.There is no changein the SCPassociated with the cold hardiness induced by chilling. Both unChilledand chilled eggsin our study had_nearly identical SCpr (ca. -27"C), and these values are quite similar to SCPspre- viously reported for overwintering gyPsy moth eggs [5-7-,25]..The well developed capacity of the eggs to supercool indicates the absenceof efficientinternal heterogeneousice-nucleating agents [26]. Since pharate larvaeof the gypsy moth do not toleratefreeziug,, the SCPindicates the minimum temperiture abovewhich the pharatelarva *iS!!!gexpected to survive,but it is clear from this study and others [e.9., 27,28]that the SCPis not a good indicator of cold hardinessand many insectssuccumb at temperaturei far above their SCP.Unlike other species,cold hardening is not associatedwith the accumulation of high concentrationsof cryo- protectants.Although glycerol was identified as the major cryoprotectant in its concentrationwas low (< 10 mM) as comParedto levels "ggt, reported^Stt.sr in other overwintering eggs [29]. proteins (heat shock pioteins) are among the pogs1b^l_efactors that *uy co.tttibute to overwintering successof the gyPsy rr,glh [9]. A_subsetof the stressproteins expressedduring heat shock, including the 75,000M. protein observedin thii sfudy, is also expres_sedat low temperafures,and the fxpressionpersists for at least6 days at 4'C. This sustainedexpression of a stressprotein at low temperaturesis especiallyremarkable because stress temperature proteinexpression -[30,31].usually ceases within a few hoursafter a high ihallenge A common mechanism may contribute to the 's responseto both high and low temperafureextremes. Low temperature stimulatesstress protein synthesisnot only in the gyPsy moth [9], but also in Drosophilamelanogaster l32l and Surcophagacrassipalpis [33]. We now rePortthat chilling plays a critical role in capacitatingdiapausing pharate larvae of the gypsy moth to respond to temperaturestress. In our experiments,stress had fi.otui" synthesiswas much more pronounced in pharatelarvae that bxperienieda period of chilling. This observationlends credenceto the idea that stressproteins contribute to cold hardiness.

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