Division of Comparative and , Society for Integrative and Comparative Biology

Cold-Shock Injury and Rapid Cold Hardening in the Flesh Fly Sarcophaga crassipalpis Author(s): Cheng-Ping Chen, David L. Denlinger and Richard E. Lee Jr. Source: Physiological Zoology, Vol. 60, No. 3 (May - Jun., 1987), pp. 297-304 Published by: University of Chicago Press . Sponsored by the Division of Comparative Physiology and Biochemistry, Society for Integrative and Comparative Biology Stable URL: http://www.jstor.org/stable/30162282 Accessed: 25-02-2016 14:44 UTC

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This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions COLD-SHOCKINJURY AND RAPIDCOLD HARDENING IN THE FLESHFLY SARCOPHAGACRASSIPALPIS'

CHENG-PINGCHEN,2 DAVID L. DENLINGER,2AND RICHARD E. LEE,JR.3 Departmentof Entomology,Ohio State University, Columbus, Ohio 43210; and Department of Zoology, MiamiUniversity at Hamilton,Hamilton, Ohio 45011 (Accepted9/16/86) Directexposure to -10 C, in the absenceof tissue ,causes high mortality in Sarcophagacrassipalpis: this result suggests that injury is due to coldshock. However, briefacclimation at 0 C enableslarvae, pupae, and pharateadults of Sarcophaga crassipalpisto survive-10 C. Chillingfor as shorta periodas 10 min enabled50% of the flies to survivea 2-h exposureto -10 C. Enhancementof cold tolerancewas linearover the firsthour of chillingat 0 C. Theoptimal temperature range eliciting the rapidacclimation response was 6-0 C, butthe effect could also be stimulatedby hightemperature (36 C). The rapid increase in coldtolerance correlates with concom- itantincreases in hemolymphosmolality and levels. This response suggests a novelrole for glycerol in protectinginsects against injury resulting from cold shock, althoughother unidentified mechanisms may be involvedin thisresponse. That both nondiapause-and -programmedflies respond to short-termchilling indicates that this rapidresponse is not partof the diapausesyndrome but probablyfunctions in eithertype of fly as an adaptationto survivebrief periods of low temperature. INTRODUCTION causing injury in cells during freezing Cold shock is a form of cellularinjury (McGrath1985). Studies observedimmediately after rapid cooling of cold hardeningin insectshave but in the absenceof ice formationin ex- generallyfocused on cold tolerance and tracellularfluids (Morris et al. 1983). This survivalabove and below the temperature phenomenonis also referredto as thermal at which spontaneousnucleation of body water shock or direct-chillinginjury. Cold shock occurs, termed "the supercooling is distinct from indirect-chillinginjury, point (SCP)"(Baust and Lee 1982). Based on this criterion,a speciesis as which occurs after long-termexposure-- categorized daysor weeks-to low temperatures(Levitt freeze-tolerantor freeze-intolerant.The 1980). The extent of cold-shockinjury in- cold-hardening process may include creaseswith higherrates of coolingand the changes in whole-body SCPs, the accu- absolutelimit of low-temperatureexposure. mulation of low molecularweight polyols Althoughnot generallyaccepted as a wide- and sugars,and the synthesisof thermal spreadcellular response to chilling,recently hysteresisfactors and ice-nucleatingagents it has been arguedthat cold shock may be (see reviewsby Baust 1981; Duman and a significant, but unrecognized, factor Horwath 1982; Zachariassen1985). To study the dynamicsof the cold-hardening process,most investigatorshave used rel- 'We appreciatethe assistanceof Dr.G. R. Needham atively long periodsof acclimationto low in determininghemolymph melting points and we thank Dr. J. J. McGrathfor his commentson the temperaturelasting more than 1 day, or manuscript.This researchwas supportedin part by more commonly, weeks (Ring 1981; Lee grantno. 8300051from USDA-CRGO to D.L.D.and and Baust 1985).But, in this studyof Sar- by the NationalScience Foundation, DCB-8517875 cophaga crassipalpis,we find that a very to R.E.L. short (10 min-2 h) exposureto low tem- 2 Presentaddress: Department of Entomology,Ohio has a in StateUniversity, 1735 Neil Avenue,Columbus, Ohio perature dramaticeffect allowing 43210. flies, even nondiapausingones, to survive 3 Presentaddress: Department of Zoology,Miami subzerotemperatures. University-Hamilton, 1601 Peck Boulevard,Ham- does not tolerate Ohio 45011. Sarcophagacrassipalpis ilton, tissuefreezing at any stageof development (Leeand 1985).The SCPin both Physiol.Zool. 60(3):297-304. 1987. Denlinger @ 1987by The Universityof Chicago.All diapausing and nondiapausingpupae is rightsreserved. 0031-935X/87/6003-8663$02.00 around -23 C, but diapausingpupae are 297

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions 298 C. CHEN, D. DENLINGER,AND R. LEE, JR. able to survivetemperatures near the SCP LOW-TEMPERATUREEXPOSURE only afterbeing in diapauseseveral weeks. Pupae used for low-temperatureexpo- Nondiapausingpupae, although they have surewere placed in testtubes (10 X 1.5cm). the same low SCP, are unable to survive Eachtreatment consisted of threereplicates temperaturesapproaching the SCP (Lee of 15-20 pupae each. After exposuresof and Denlinger1985). variousdurations to chillingtemperatures This studyfocuses mainly on the pharate (0 and/or -5 + 1 C), pupaewere exposed adultstage of nondiapausingflesh flies and to temperaturesof -10 C or below using a describesthe effectof briefperiods of chill- LaudaRMT-20 (Brinkmann) low-temper- ing at 0 C on enhancingthe fly's capacity ature bath filled with water and ethylene to surviveat lower temperatures(-10 C). glycol (1:1).All pupaewere then returned Several other developmental stages, in- to 26 C until adultemergence. cludingflies programmed for diapause,are also examined.We test the possibilitythat CRYOPROTECTANTDETERMINATION cryoprotectantlevels may riserapidly in re- Low molecular were an- to ex- weightpolyols sponse short-term,low-temperature alyzed by high performanceliquid chro- posure. matography (Waters Associates) as de- MATERIALAND METHODS scribedby Lee et al. (1983). Sampleswere storedin a freezerat -40 C beforeanalysis. INSECTREARING For each extraction,two specimenswere A colony of the flesh fly, Sarcophaga weighed and homogenized in 3 ml of crassipalpisMacquart, was maintainedin methanol in a Teflon-glasstissue homog- the laboratoryas describedby Denlinger enizer for two 20-s intervals.The homog- (1972). Parentaladults were reared at 25 C enizer was rinsedwith 2 ml of methanol with either a diapause-inducingphotope- and the samplecentrifuged at 2,000 g for riod (12L:12D) or a nondiapausephoto- 5 min. The supernatantwas transferredto period (15L:9D). Larvaeand pupae were a clean sample tube, and the pellet was kepteither at 20 or 25 C underthe maternal reextractedtwo more times with 3 ml of photophase.Short-day conditions at 20 C methanol. The pooled supernatantwas producea high incidence(>95%) of pupal forcedthrough a prewashed(2 ml methanol diapause.The developmentalstatus of each and 2 ml distilledwater) Sep-Pak C18 car- pupa was determinedby removingthe an- tridge and evaporatedto drynessusing a teriorportion of the pupariumand looking Reacti-Vap evaporator (Price Chemical forsigns of antennalformation and the eye- Co.) with low heat and compressedair for pigmentation characteristicsof pharate 3 h. The samplewas then resuspendedin adult development (Fraenkeland Hsiao a 0.5-ml ethanol:watermixture (1:1) and 1968). filteredthrough a 0.22-rgmfilter and ana- concentrationswere ex- DEVELOPMENTALSTAGES lyzed. Glycerol pressedin mM units based on water-con- At 20 C, larvaefeed forabout 7 daysand tent data (Adedokunand Denlinger1985) then leave the food as third instar larvae reportedfor correspondingdevelopmental and enter a wanderingphase that lasts 4 stagesof the same species. days for nondiapause-destinedlarvae and 6 days for diapause-destinedlarvae. Pupa- tion occurs4 days afterpupariation, and, MELTINGPOINT DETERMINATION if diapause intercedes, development is Hemolymphmelting points were mea- halted at the stage of the phanerocephalic suredwith a nanoliterosmometer (Clifton pupa.In nondiapausepupae, pigmentation TechnicalPhysics) using the method de- is visible 14 days after pupariation, and scribedby Frickand Sauer(1973). Standard adults emerge aroundday 21. At 25, pu- osmolarconcentrations and distilledwater pariationoccurs 4 daysearlier, and the time wereused with each sample platform. Dur- frompupariation to adulteclosion is 9 days ing the melting process,temperature was less. Diapausecan be maintainedat 20 C slowlyincreased until only a single crystal for more than 120 days,but some individ- was visible. Readingsin mosmolarswere uals startbreaking diapause after 60 days. transformedto melting points using a

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions COLD SHOCK AND COLD HARDENING IN FLIES 299 S 250 1. 190 5o I 2, io1 90 250 60 I 10,o 1 250 0 1 60

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0 4 8 12 16 0 20 40 60 80 100 Exposure Time (hrs) N % Emergence FIG.1.-Effect of short-termchilling on cold toleranceof Sarcophagacrassipalpis. All sampleswere reared underthe nondiapauseprogram at 15L:9Dand 25 C and testedas pharateadults (red-eye stage). After exposure to -10 C for 2 h, survivorshipto the adultstage is expressedas meank SE of at leastthree replicates consisting of 20 flieseach. meltingpoint depressionof 1.86 C per 1.0 C, pharateadults were exposedto 0 C for osmole. differentintervals and then transferredto -10 C for 2 h. A 10-min to 0 C RESULTS exposure was sufficientto permit 50%of the flies to EFFECTSOF SHORT-TIMECHILLING ON SURVIVAL survive-10 C, and exposureof 1 h or more When nondiapausepharate adults (red- at 0 C permittednearly all flies to survive eye stage)were transferred directly from 25 -10 C (fig.2). The increasein survivorship to -10 C for 2 h, very few surviveduntil was nearlylinear for the first30 min of ex- adult emergence(fig. 1). In contrast,flies posure to 0 C. The relationshipbetween were highly tolerantof a 2-h exposureto chilling time and log increasein survival -10 C if they firstexperienced a 2-h accli- over the first 60 min is defined by the mation period of 0 C. Some flies (38%) could even toleratea 2-h exposureto -13 C if they were first acclimatedby 2-h ex- posureto 0, -5, and -10 C. However,short periodsof chillingdid not enable the flies to survivea temperatureof -17 C. 0 The dataof figure1 indicatethat survival at -10 C is enhancedonly by short-term chillingthat precedesexposure to -10 C, ratherthan exposureto -5 or 0 C, which occursafter the -10 C treatment.Thus, a ao short-termwarm-up following exposure to 30 so 90 120 -10 C cannot reducecold injury. Duration of Exposure to 00 C (min) FIG.2.-The relationshipbetween cold tolerance MINIMUMEFFECTIVE DURATION OF CHILLING for 2 h at -10 C and durationof priorexposure to 0 C. Larvaeand pupaewere maintained at 20 C and To determinethe minimumduration of testedas pharateadults (red-eye stage). X I SE, sur- chillingrequired to enhancesurvival at -10 vivorshipof threereplicates containing 20 flieseach.

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions 300 C. CHEN, D. DENLINGER, AND R. LEE, JR. 100 was tested: pharateadults (red-eyestage) rearedat 25 C wereexposed for 2 h to tem- peraturesfrom 36 to -7 C and then trans- 75 ferredto -10 C for 2 h. A temperature rangebetween 6 and 0 C was most effective from 12 to 50 (fig. 3). Temperaturesranging E 30 C had little or no effect in enhancing survivalat -10 C, but, surprisingly,a 2-h to 36 C enabled40% of the flies 25 exposure to survivea 2-h exposureat -10 C.

EFFECTOF GRADUAL COOLING 01- 35 30 25 20 15 10 5 0 -5 -10 Temperature(rC) To test the effectof gradualcooling ver- sus an abrupttemperature drop, flies were FIG.3.--Effects of differenttemperatures (2-h ex- cooled from 25 to -10 C posure)on thecapacity of pharateadults (red-eye stage) gradually(0.54 of Sarcophagacrassipalpis to survivea 2-h exposure C/min)or in abruptsteps (fig. 4). Only4.5% to -10 C. Flies were rearedunder the nondiapause of the flies surviveda 2-h exposureto -10 programat 15L:9D,25 C priorto the experiment.X C followinga gradualtemperature decrease + SE, survivorshipof threereplicates containing 15 flieseach. from 25 to -10 C. Survivalat -10 C was directlyrelated to the absoluteamount of previousexposure to 0 C and was not fur- regressionequation y = 0.654 - 0.009x; ther enhancedby a gradualtransition. r = -0.978. DEVELOPMENTALEFFECTS OPTIMALCHILLING TEMPERATURE The aboveexperiments focused on phar- The above experimentused 0 C as the ate adults(red-eye stage) reared under non- stimulantfor enhancingsurvival at -10 C. diapauseconditions (25 C; 15L:9D).In this In this experiment,a rangeof temperatures experiment,we evaluatedthe effectof short-

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0 20 40 60 12 0 20 40 60 80 100 minutes hrs Exposuretime N % Emergence FIG.4.-Effects of abruptand gradualchilling on cold tolerance(2-h exposureto -10 C) in pharateadults (red-eyestage) reared at 15L:9D,25 C. X + SE, survivorshipof threereplicates. Gradual chilling represented by converginglines.

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions COLD SHOCK AND COLD HARDENING IN FLIES 301 chilling at 0 C consistentlyenhanced the abilityof nondiapauseflies to survivea 2- h exposureto -10 C (fig. 5A).The effectof chilling was most dramaticin increasing survivorshipof third instarlarvae and ad- vanced stages of pharate adult develop- ment. The pupal stage(about 2 days after andthe 25 pupariation) earlyphase of pharate adultdevelopment are more tolerantof di- rect exposureto -10 C, but, even during a . A these stages, survivorshipis greatly en-

E 100 hancedby a 2-h pretreatmentat 0 C. B Among flies programmedfor diapause " (fig. 5B), a 2-h exposureto 0 C again en- 75 hanced survivalat -10 C. The effect was most apparentprior to and shortly after pupariation.Within 4-5 days afterpupar- o 2 h exposure to 0'C * unchilled iation,the fly is in the phanerocephalicpu- pal stage characteristicof diapause,and, 25 from this time onward,diapausing pupae were alreadyquite cold tolerantand fully capableof survivingat -10 C withoutpre- FL WL 0 2 4 6 8 10 12 14 A treatmentat 0 C (Leeand Denlinger1985). Days AfterPupariation CHANGESIN GLYCEROLCONCENTRATIONS AND FIG.5.-Developmental changesin the abilityof A, nondiapause-and B, diapause-programmedflies to re- HEMOLYMPHOSMOLALITY spondto chillingfor 2 h at 0 C. Priorto chilling,flies to be the weremaintained at 20 C. Followingchilling, flies were Glycerol appears major low exposedto -10 C for 2 h and X c SE survivorship molecularweight cryoprotectantused by was recordedfor threereplicates of 15-20 flies each. Sarcophaga(R. E. Leeand D. L. Denlinger, FL = feeding larvae, WL = wandering larvae. unpublisheddata). A 2-h exposureto 0 C was adequateto significantlyelevate glyc- termchilling on otherdevelopmental stages erol levels 2-3-fold comparedto unchilled of nondiapauseflies and also on flies pro- controlsfor all developmentalstages tested grammed for diapause. A 2-h period of (table 1).

TABLE1

EFFECTOF SHORT-TERMCHILLING FOR 2 h AT 0 C ON TISSUECONCENTRATIONS OF GLYCEROL INSarcophaga crassipalpis

GLYCEROLCONCENTRATIONS

ig/mg Wet Weight mM DEVELOPMENTALSTAGES REPLICATES(N) (x+ SE) (X+ SE) Wanderinglarvae (long day): No chilling 3 1.2p .4 18.2g 3.7 2 h at 0 C ...... 3 2.4 .2 ...... 43.0 + .2 Wanderinglarvae (short day): No chilling 6 .8 .2 m ...... + 13.7 4.9 2 h at 0 C 2.4 .4 h ...... 3 + 42.1 6.4 Pharateadults (long day): No chilling 9 ...... 1.7k.1 28.2 + 2.7 2 h at 0 C 9 3.4 q ...... +.1 81.4 3.0 NOTE.-Larvaewere reared at 20 C at eitherlong-day condition (15L:9D, nondiapause program) or short-day condition(I12L: 12D, diapause program). Differences within each couplet are significant (Student's t-test, P < .05). Two fliesin each replicate.

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions 302 C. CHEN, D. DENLINGER,AND R. LEE, JR. A 2- or 3-h exposureto 0 C also resulted apparentinstance of cold shockin the grain in a significantincrease of hemolymphos- weevil, Calandra granaria. Additional molalityin pharateadults (table 2). A com- study is requiredto determinehow com- parisonof tables I and 2 suggeststhat the mon cold shockis among . increasein hemolymphosmolality may be The generallyaccepted hypothesis for the accountedfor by the increasein glycerol mechanismof injury resultingfrom cold concentrationsfor chilledpharate adults. shock is the inductionof phasetransitions in membranelipids and the subsequentloss DISCUSSION of membranepermeability (Morris et al. Short-termexposure to 0 C stimulates 1983;Quinn 1985).Cryoprotective agents Sarcophagacrassipalpis to undergoa rapid such as glycerol may alter the nature of physiologicaladjustment that enablesthe these phase transitions,stabilize relation- fly to surviveshort-term exposure to -10 ships between bilayer and nonbilayer C. A 10-minexposure to 0 C was adequate forminglipids, and thus preventthe redis- to allow about 50%of the flies to survive tribution and segregationof membrane -10 C for 2 h. We have demonstratedthat componentsupon thawing(Quinn 1985). this mechanismcan operatein larvae,pu- Injurymay also be a result of membrane pae, and pharateadults of both nondia- failure owing to thermoelastic pause-and diapause-programmedflies. Al- (McGrath1984). though nondiapauseflies cannot achieve In insects,the processof cold hardening the same level of cold tolerance as dia- is often associatedwith the accumulation pausingpupae, it is clear from this study of glycerol(Zachariassen 1985). Although that, if properlyacclimated, they too can a varietyof mechanismsof protectiveaction toleratesubzero temperatures. have been suggestedfor glycerol,it is gen- Rapidcooling to -10 C in S. crassipalpis erallyagreed that it primarilyfunctions to resultsin highmortality (fig. 1), despitethe either:(1) depressthe temperatureof het- factthat tissue freezing does not occuruntil erogeneousice nucleation(i.e., the SCP)in -23 C (Lee and Denlinger 1985). Injury body tissues or (2) preventinjury to cells after rapid cooling, but without tissue after the formationof ice in extracellular freezing,indicates that mortalitywas due spaces. In the formercategory, high con- to cold shock.In an extensivebibliography, centrationsof glyceroland, sometimes,of Morrisand Watson(1984) documentcold otherpolyhydric alcohols and sugarsappear shock in a wide range of ,in- to function as low molecularweight anti- cluding bacteria,yeast, algae, fungi, pro- freezecompounds that increase the capacity tozoa,higher plants, fish, spermatozoa, and for the supercoolingof body water and, mammalian somatic cells and embryos. thereby,decrease the likelihood of tissue Although no specific referencesto cold freezing. Alternatively,in freeze-tolerant shockin insectswere cited, it is well known species,these compounds serve as cryopro- thatsome insectsdie at temperaturesabove tective agentsprotecting cells as ice forms their supercoolingpoints (Ring 1980). in extracellularspaces. In a discussionof Ushatinskaya'swork, Rapidcooling from 25 to - 10 C resulted Solomonand Adamson(1955) describean in >90% mortalityin pharateadults of S.

TABLE2

CHANGES IN OSMOLALITYAND HEMOLYMPHMELTING POINTS IN PHARATEADULTS (RED-EYESTAGE) OF Sarcophaga crassipalpis IN RESPONSETO CHILLING AT 0 C

HemolymphOsmolality HemolymphMelting Point Treatment mOSM(X+ SE) oC (Xx SE)

No chilling ... 308.7 s 6.6 -.574 o .012 2 h at 0 C .... 346.3n 4.5 -.644 b .008 3 h at 0 C .... 344.3e 3.5 -.641 k.007

NOTE.-Larvaeand pupae were reared at 15L:9D;20 C. Differencesbetween chilled and unchilledflies are significant (Student's t-test, P < .05). EachN = 3.

This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions COLD SHOCK AND COLD HARDENING IN FLIES 303 crassipalpis,while an intervalof only 2 h temperature-dependentproperties of the at 0 C priorto exposureto -10 C yielded two enzymesresult in a slowbut continuous >90%survival (fig. 1). Duringthe same 2- accumulationof glycerolat low tempera- h intervalat 0 C, glycerollevels increased ture. morethan 2.5 timesrelative to controls(ta- Interestingly,the survivorshipdata show ble 1).Most experiments report a significant that a 2-h exposureto 36 C also can en- accumulationof glyceroland polyolsafter hance survivalat -10 C. It thus appears severaldays or weeksof acclimation(Baust that high-temperatureexposure can elicit 1982; Rojas et al. 1983;Nordin, Cui, and the same biologicaleffect as temperatures Yin 1984),but our resultsdemonstrate that around0 C, althoughthe mechanismis not glycerolsynthesis is a very rapidresponse necessarilythe same. High temperatureis to low temperaturein flesh flies. The con- known to stimulate polyol formation in comitantincrease of glycerolwith the rapid larvaeof anotherDipteran, Callitroga ma- enhancementof cold toleranceat -10 C cellaria(Meyer 1978). suggeststhe possible existence of a third Acclimationto low temperaturegener- categoryof protectiveaction for glycerol: ally requiresexposure periods lasting days glycerol protects insects against injury or weeks(Colhoun 1960). However, Meats causedby cold shock. But, additionalcel- (1973) reportedrapid acclimation with re- lular events such as membranereorgani- spect to thresholdsfor torporand flightin zation or the productionof specific pro- the fly,Dacus tryoni.Maximal levels of ac- teins, comparableto those associatedwith climation were observedeven at cooling the heat-shockresponse, may be involved. ratesof 1 C/min. Fly survivaldata suggest that the optimal This short-termacclimation mechanism temperaturefor this short-termacclimation may be of criticalecological significance. It rangesfrom 6 to 0 C. This is similarto the implies that stages other than diapausing optimalrange for the inductionof the syn- pupae can rapidlyenhance low-tempera- thesisof cryoprotectivecompounds in other ture tolerance.Although only diapausing insects (Baust 1982). One of the key en- pupae can toleratethe prolongedperiods zymes, glycogen phosphorylase has its of low temperaturecharacteristic of highestactivity between 4 and 0 C in pupae (Adedokunand Denlinger 1984; Lee and of the silk moth,Hyalophora cecropia (Zie- Denlinger1985), nondiapausingindividu- gler and Wyatt 1975; Ziegleret al. 1979). als are not likely to be killed by an occa- However,two enzymesystems are involved sionalnight in autumnor earlyspring when in regulatingthe response:phosphorylase temperaturesdrop to an unseasonallow. In kinase and phosphorylase phosphatase fact, our data suggestthat the extremely (Hayakawaand Chino 1983; Hayakawa rapidacclimation observed in S. crassipal- 1985).Activity of phosphorylasekinase re- pis may allow this species to "instanta- mainshigh at low temperature,while phos- neously"enhance cold toleranceas it tracks phorylasephosphatase shows very little ac- decreasesin its environmentaltempera- tivity near 0 C. Thus, in silk moths, the tures.

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This content downloaded from 134.53.236.18 on Thu, 25 Feb 2016 14:44:30 UTC All use subject to JSTOR Terms and Conditions 304 C. CHEN, D. DENLINGER,AND R. LEE, JR.

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