506 Inoculation triggers freezing at high subzero temperatures in a freeze-tolerant frog (Rana sylvatica) and ( solidaginis)

hcr R. LnyNr, Jn.l OhioUniversity - Belmont,45 425National St.Clairsville, OH 43950, U.S.A. .Road, Departmentof Biotogy,Nazareth Cotlege, f,ZT, Uor, orrnue, Rochester,NY 14610,q.S.A. Rrcseno E. LBe, Jn. Departmentof Zoology,Miami Universit:v,160l PeckBoulevard,Hamilton, OH 45011,U.S.A. AND JeNsr L. Hue,Nc Department of Biology, WheelingCollege, Wheeling, WV 26003, U.S.A. ReceivedApril 4, 1989

Levxe, J. R., Jn., Lee, R. 8., Jn., and HunNc, J. L. 1990.Inoculation triggers freezing at high subzerotemperatures in a freeze-tolerantfrog (Rana sylvatica) and insdct (Eurosta solidaginis). Can. J. Zool. 68: 506-510. Freezing of is often portrayed to begin after they supercoolseveral degreesCelsius below the melting point of body fluids. This suppositionis basedon laboratory protocol that usually exposesanimals only to dry air during freezing episodes, whereas, in nature, animals may be in direct contact with external ice or snow at temperaturesabove the supercooling point of their body fluids. This raisesthe possibility that ice nucleationmay occur acrossthe epirheliumor cuticle, causingthe freezing of body fluids. We testedthis possibility in two freeze-tolerantanimals, the wood frog, Rana sylvatica, and the goldenrod gall , Eurosta solidaginis. Frogs remained supercooledfor 3 h at - 1.5 to -2.0"C when kept on an unfrozen surface;however, they beganto freezein lessthan 30 s after coming in direct contactwith ice crystals. Seedingoccurred in lessthan I min acrossisolated patchesof frog skin held in a modified Ussing chamber. Similarly, externally moistenedlarvae of E. solidaginis froze at higher temPerailres than dry larvae. Likewise, within galls containing a relatively high water content (65.87o), larvae froze at higher temperaturesthan onesfound in drier galls (l9.6Vo). Therefore, animals may freezeat high subzerotemperatures, at or nearthe melting point of their body fluids, owing to transepithelialice inoculation. In E. solidaginrs,exposure to sufficient moisture to trigger inoculation declinesas winter approaches;thus, this avenuefor freezing seemslimited to autumn, when plant galls have a high water content. This study further emphasizesthe needto use care in extrapolating laboratory-determinedsupercooling points as an approximationof the responseof animals in the field.

L,lvnr, J. R., Jn., Lee, R. E., Jn., et HUANG,J. L. 1990.Inoculation triggers freezing at high subzerotemperatures in a freeze-tolerantfrog(Rana sylvatica) and insect (Eurosta solidaginis). Can. J. Zool.68: 506-510. Il est gdndralementreconnu que la cong6lationd'un animalcommence lorsqu'il atteintson point de surfusion,qui est de plusieursdegrds Celsius au-dessous du point de fusionde sesfluides corporels. Cette supposition est bas€e sur le protocole expdrimentalau cours duquel les animaux sont ordinairement exposds seulement d deI'air sec,alors qu'en nature ils sontsouvent en contactdirect avec de la glaceou de la neige,e destemperatures sup6rieures au point de surfusion de leursfluides corporels. Celanous amine i envisagerla possibilit€qu'il peutse former des noyaux de glace dans l'€pithdlium ou la cuticule,entrainant le geldes fluides corporels. Nous avons mis cette hypothdse i l'6preuve chez deux animaux qui peuvent toldrer le gel, la Grenouille desbois, Rana sylvatica, et la Galledes verges d'or , Eurostasolidaginis . Les grenouilles sont rest6es en surfusiondurant 3 h A -1,5 a 2,0"Csur une surface non gel6e;cependant, elles ont commenc€ i geleren moinsde 30 s lorsqu'ellessont entr6es en contactdirect avec des cristaux de glace.Des cristaux ont commencd d se former en moins de I min surdes morceaux isol€s de peaude grenouille gard6s dans une enceinte de Ussing modifide. De m€me, des larves humidifi6es d'8. solidaginisontgel€ i des temp€raturesplus €lev€esque deslarves sdches. A I'intdrieurde gallesi contenuhydrique relativement €lev6 (65,87o), les larvesont gel6i destempdratures plus dlev6es que les larves rouvdes i I'intdrieurdegalles plus sdches (19,6%). Les animaux peuventdonc geler i destemperratures sous-z6ro 6levdes, au point ou auxenVirons du point de fusion de leurs fluides corporels, parinoculation transdpith6liale de glace.Chez E . solidaginis,l'exposition i unehumidit6 suffisamment dlevde pour d6clencher I'inoculationde glace diminue i mesureque I'hiver approcheet cettevoie de cong6lationsemble donc limit6e i I'automne,au momento! lesgalles des plantes contiennent une grande quantitd d'eau. Cette €tude nous incite i rdit€rerla miseen garde contre I'habituded'extrapoler les donn€es de laboratoiresur les points de surfusionpour pr€dire les r€actions des animaux en nature. [Traduitpar la revue] Introduction fluids after modest supercoolingto high subzerotemperatures Freezing is lethal to most animals; however, a number of and, in some 'they are known to be proteins or lipoPro' (Duman invertebraiesand a few vertebratesregularly survive extensive teins which are producedseasonally et al. 1985)' the goldenrod gall fly, Eurosta ice formation in their body fluids durilngthi winter. Efforts to ._Frequently, solidaginis define how and wherefreezing first begiis in theseanimals have (Diptera, ), and the vood frog, Rana sylvatica, are focused on the role of internlalnucle-ating agents (Bale ercl. used.as mod_elsfor studying freeze tolerance in insects and 1989; Baust and Rojas 1985; Duman t-981-;Somme 1978). vertebrates(Baust and Lee l98l; Baust 1986;Layne and Lee 1987;.Storey and Storey 1988). Earosta solidaginis Theseagentspromoteicecrystalformationinextracellularbody- lantae overwinter in stemgalls that project abovethe snow where they l;uho;*hom all conespondenceshould be sent ar the following may be exposed lo extremlly low temperatures during thl address:Department of Biotogy,Nazareth Colle ge,4245 East Avenue, winter (Uhler 1951).In contrast, wood frogs overwinter nearor Rochester,NY 14610,U.S.A. at the soil surfaceunder leaf litter and snow on the forestfloor LAYNE ET AL 507 (Schmid1982). Correspondingly, winrer_conditioned, E. solid_ 2 cm thick styrofoamplug.filled the opening aginislarvae regularly survive _ of the beakerro prevent temperaturesbelow 20oC(Lee excessivetreatexchange^with room and Lewis air. Ethyleneglycol circulatedfrom 1985) and somepopulations from northern a NeslabRTE 2l0A refrigerated Canada bath rhroughtfiebeakerlacket. Tfre enduretemperaturesto -55;C(Salt 1957), beaker compartmenr *h...u. the lowei was-held at equilibriJm i.rnp..u,u... ranging limit of survival for R. _6 _g"C from -1.5 to -2.0"C s,-lvaticcis near to (Schmid andpreliminary t,uf,,to*.0 that neitherthe 1982;Storey 1985). filte.r paper nor the frogs spontaneously froze within 3 h. After In equilibrating for I h. a piece-of the laboratory, many freeze_tolerantanimals supercool ice was i.opp.a only onto the filter paper (not directly onro rhe frog) severaldegrees below themelting point of theirbody fluids. through'a small'op.ning in ih. For styrofoam cover, causingwater ex1mpl9,gall fly larvaecommonly supercool _6 irithe moiitened ntt.. pup.. 6 fr."r. - to between within seconds.It was evident that and 10oCprior to theonset the onsetof freezing'for riresefroli of ice nucliation (Baleet al. l9g9t; was due to contact with Baust ice and not to spontaneousinternal ice er al.1979; Baustand Lee l9gl, l9g2;Lee andLewii nucleation. 1985;Morrissey and Baust 1976). Freeze-tolerant supercooling points were frogs typic_ , assessedseparately for insect larvae and ally supercool2 to 4"C below the their galls. A thermocouple melting point of the blood was placed uguinriit OoOyof each larva prior to spontaneousfreezing (Layne " un-d'L". 19g7, l9g9; yru,pp"q.togetrrerwith a single layer of op.. a Lotshaw mermocouplef-.*Y|..::r: 1977; MacArthur and Dandy, l9g2; Schmld tqSZ; was placed,betweenthe two halvis of tt. op"n.j Storey from which the larvae !At, 1984, 1985;Storey and Storey 19g6, l9g7) and they had beenremoved unJin.l*o halveswere put tape..Gails can remain supercooledbetween _2 to _3"C for up ana inr..t, *.r" uttowedto equilibrate to to 20 h ?*Il:t.,l:::ithru L ln rneretngerated (Layneand Lee 1987). bath.The insectsand galls were cooled at a rate of0.8 to 0.9"C/min An alternatemechanism until spontaneousfreeziig occurred. for initiating freezingin the body To demonstrateinoculation fluids is by inoculation acrossthe integ'umentof f.ogs, patches throughtheir extemal surfaies.Materials of dorsal and ventral skin were like removed from recently sacrificed adult soil and plant tissuesmay be moist and freeze frogsand held in a modified at tem_ Ussingchamber (ielman anOMiller 197I peratureswell abovethe supercooling Tumer et al. 1985). ; limits of the body fluids A sectionof iirin fZ cm ln Jiameter)was cenrered of their respectiveinhabitants. in the apernue berween Con6ct with externalice has two identical *eff s ta anJi) containing a 0. I b:"1^r!^o*_1to trigger M NaCl solution. The jacket freezing in some insect species(Bale er of each well ieceived coolant from the refrigerated bath al. 1989;Humble and Ring 1985;Salt 1950, i963; Shimada and the temperature of each well was monitoreJ and Riihimaa separately.A small piece of ice was 1988; Zachariassenl9g5). However, little em_ introduced into well A in contact phasis with the extemal surface of the skin has b-eenplaced on inoculative freezing as a pnmary to seedits supercooledsolution once thermal equilibriumwas _b.g.C mechanismfor triggering freezing in reachedat either or _1.5"C- nature. Temperature was recorded This study.documents in the opposing *"ll i for up to 5 min the occurrenceof inoculativefreezing thereafter. in both E. solidagims larvae and R. sylvatica.We also The. supercooling limit was determined for soil samples near demonstratethat seeding can occur across werwintering the integument of sites used by R. sylvatica in Ontario Co., New york. frogs. The top 2 cm of soil was used for these tests. nact sampte *as packagedinto approximately Materials 50 g.units_andptu.eO in pfurii. p.trl and methods dishes.A thermocouple wus inr"rt.id into tt...,itr" of .u.t sumpt".unA Subadultwood frogs wereobtained pennsyl- temperature logged from FranklinCo., _was on the data recorder. Dishes were then placed vania(U.S.A.), in lateNovember, into the refrigerated 19g7,and a$ult wood frogs wire bath and allowed to ..u.f, an equilibrium collectedfrom Athens Co., Ohio(U.S.A.), temperatureof 5'C. _5oC in earlyVtarch, t9-SA. eI The bath was resetto ana tne sampfeswere frogs were held at 3'C for Z to weeks allowedto cool at 5 and fastedprior to a rateof 0.5 to 1.O.C/minun,iifontun"ous freezing experimertation.During mid to late Octoberand Novembei,l9gg, began. glantgalls containinggoldenrod gall fly Mean valrresare reponed laryaewere collected from along with I SE. Analysisof main effects MonroeCo., New York. All experiments ror" groups oI means on larvaewere performed were done using two_wayANOVA. and multiple immediatelyafter returning mean to the laboratory. comparisonswere made using Tukey teits. Statistical Thenatural freezing of gail sonsof individual . . fly rarvaewai recordedat thecolrecting paired setsof meins *.." rnuO"uring r_tests."o_fii_ siteon october3l followingan overnight freeze (- 6.r'c). GailswerE openedbetween 0900 andr000 and inspected for evidenceof icein the Results planttissues and larvae.This was a visual inspectionand thusonly E. solidaginislawae extensivefreezing was detected. . .Both andR. sylvaticasupercooledin the laboratory when permitted Inoculativefreezing was initia-tedin E. sotidaginislarvae using to do sb. Wood irogs remained procedures unfrozenat temperarures _ comparableto thoseof Salt(1963). Lariae wereremoved between 1.5and _Z.O.t f";; b"; fromsome plant galls and were placed aspermitted in on water-saturatedfilter paper or this study (up to 3 h). The water_saturatedfilter on the.bottom u dry plastic paper 9f- petri dish. Anotherset of galli was at the bottom of ttre.yacteieAU"ut".-ufro supercooled opened,checked for thepresence of live larvae,and resealed during this period; i,ith tape. however, spontaneousfreezing occurred in Isolatedlarvae and gails werethen placedinto a dry containerand poth th9 'ihe _frogsand the filtei paper if the temperature was immersedin a NeslabRTE 2l0A refrigeratedbath. remperature lowered. Sponraneousfriillg off . solidaginis iu*ue geno_ insidethe well was heldbetween -5.2 and -6.2"C. A final set -9.9 of ally occurredat about l). Larva"e larvaewas kept on water-saturatedfrlter paperat -2.9 to -3.1.C. (!!g. usually.oild b" keptunfrozen at -5.2 to -6.2"Cfor After24 h, larvaewere examined for thepreience of ice by probing. 24 h if they werekept on a -e dry surface (seebelow). .Similarprocedures were used to initiaie freezingin livi irogs. The presenceof ice was-determined subadultfrog wasplaced on a pieceofwater_suturut.i filt..pup"r"ut th. -_ by probing the larvae. Unfrozen bottomof a 500-mLjacketed beaker. A cup_shapedwiri cagewas larvae had flexible bodies even ui ruUr"ro rempera- placedove_r each frog tures,.although anda thermocouplewas placed against the frog,s they showed.no mobility in response to dorsalsurface. Additionai prodding. thermocouplesweri positionedon thefilter Frozenlarvae had rigid bodies,unA,upiA dissection paperand in theair I cm abovethe bottom of a larva ofthe beaker.Temperatures revealedconsiderablJ amounts of internalice. werecompiled wirh an Omega -2.9 When RD_106multichannel recorder which fust held at to -3.loC, the l*a. cycledtkough six unJ*arer-saturated channelsevery 16 s. Thus,the appearance ofthe filter paperdid not freeze, exothermin each evenafter 24 h. Oncea smallpiece of frog wasaveraged on thebasis of tireserntervals. A icewas placed into thepetri dish, thefilter paperLeganto freeze cAN. J. ZOOL.VOL.68. 1990

80

860 6o c o Eoo o lrJ IE o f 620 |'-1 DORSALSKIN = q E --- FROGNo.l UJ -- November o- FROGNo.2 = UJ 120 l- i0{, g -2 880 N 0 200 400 o ti 60 o s40 0 Jzo6

0 () October November o UJ E o F E VENTRAL SKIN o a E lu ---*r_ FROG No.l CD o- --.- eo = FROG No.Z o o UJ o F CL o -2

October l,lovember s00 1000 Ftc. l. Frequencyhistogram of (A) the water contentof plant galls TIME (s) andlarvae during Octoberand November, (B) the percentageof Euosta solidaginislarvae frozenfollowing 24hat -5.2to -6.2"C, and (C) Ftc. 2. Temperatureprofile for Ussingchamber showing hanscuta- supercooling points for eight larvae and their galls in October and neousice nucleationacross dorsal and ventral skin of the wood frog, Rana November. In (B), the total number of larvaeused in eachtest group is sylvatica. given above each histogram. The vertical bars given with histograms for water content and supercooling point denote I SE. Note that no flexible bodies and showed no color change, and these were larvae from the November group froze when held on the dry surface. judged to be unfrozen or only partially frozen. External ice similarly initiated freezing of frogs. For exam- immediately and all larvae were solidly frozen within 24 h. ple, dropping a small ice crystal onto the supercooled,wet filter Spontaneous freezing of the filter paper and nearly all insects paper in the seeding chamber led to growth of ice through the --5.2to occurredduring thefrst 24 h rial at -6.2'C (Fig. l). In wet filter paper.Each frog subsequentlyshowed a suddenrise in contrast,only a few larvaespontaneously froze at this tempera- temperature that was detected by a thermocouple positioned ture when kept on a dry surface for 24 h. Thesetrends did not against its dorsal surface. The air temperature at the sameheight differ between the October and November test groups. Like- of each frog's dorsum rose slightly in responseto the heat of wise, their supercoolinglimits showedno significant (P > 0.05; fusion releasedby the moist filter paper and usually returned tci two-way eNova) difference between these2 months (Fig. 1). between -1.0 and -1.5'C after 2 or 3 min. The substantial Inoculative freezingoccurred in E. solidaginrsthat were kept changes in body temperature were thus due to the heat of fusion - - in plant galls at 5 .2 to 6.2"C (Fig. 1). This capacitydeclined releasedby the frogs and not from the water freezing in the filter dramatically betweenOctober and November, a fact which was paper. Frogs began freezing an averageof 14-30 s (N = 6) paralleledby a significant(lD < 0.001;Tukeytest) decline in the following the seedingevent; the longest time for the appqrance water content of the galls. However, no significant (P > 0.05; of an exothenn was a 34-50 s interval. Once freezing two-way exove) changein the supercoolingpoint of plant galls commenced, the body temperature of each frog rose to between occurredduring this period (Fig. l). Field observarionsrevealed -0.3 and -0.5"C. Indeed, after 2 h, frogs had stiff limbs and that all galls were frozen on October 3l following a freezethat rigid abdomens,which is indicative of extensive freezing of -6.1'C. attained Five of the l0 larvaeobserved had accumu- their body fluids. lated high ice contents. The remaining larvae had relatively Experimens using the modified Ussing chamber revealed LAYNE ET AL thatinoculative freezingoccurred rapidly acrossthe integument Hyla versicolor in Minnesota with its supercoolirt'slimit of wood frogs. This was evidenced by a sharp rise in (-2.1'C). Houever.freezing episodes could bccur u, J*ly us temperature caused by the freezing of the water in well B Novemberand extend into mostof Marchif inoculativefreeiing immediatelyfollowing the introductionof ice crystalsinto well occurswhen soil temperaturesare -0.5.C or lower. (Fig. A 2). The temperatureof well ,B invariably rose to a External nucleationin E. solidaginissimilarly requiresthat plateau less than I min after seeding of well A. Visual moisture in surroundingplant tissuesfreezes at highei tempera- revealed inspections that, afteronly 5 min, substantialamounts turesthan individuai larvae. The critical temperaturerange here of ice had formed in -1.2 -3.0'C j6.0 both wells. No difference was apparent is between to (Baustand Lee l9g1) and to the seeding - between timesfor ventral and dorsalskin (Fig. 2). 10.0'C, andour dataindicate that galls commence freezing at The overwintering substratesfor R. sylvatica (soii) and E. temperatureswell under the supercoolinglimit of intact larvae. (plant galls) solidaginis underwent spontaneousfreezing at Moreover, supercoolingpoints reported here (Fig. l) for the higher subzero temperaturesthan did their respectiveanimal plant galls are low sincegalls were cooled faster than would be occupants(Fig. l). Supercooling limits weri not directly expectedunder naturalconditions and frost is likety to form on determinedfor frogs, but individual R. sylvatica could be held the surfacesof galls in the field, which may -1.5 -2.0"C then trigger freezing between and for 3 h without spontaneously at higher ternperatures. freezing. The lowest single supercoolingpoint for our soil For some insects,in

integumentpermits rapid seedingby external ice, as evidenced avoidingedge damage in studiesof frog skin.Science (Washington, by our experiments with the Ussing chamber. This was not D.C.),173: 146-148. totally unexpectedsince most amphibians have highly porous Huunlr, L. M., andfu Nc, R. A. I 985.Inoculative freezing of a larval skins; however, it cannot be conclusively stated from the parasitoidwithin its host.Cryo Lett. 6: 59-66. Levxr, J. R., present data that the integument is the specific route for Jn., andLr,e, R. E. , Jn. 1987. Freezetolerance and the dynamicsof ice formationin (Rana inoculation in live frogs. Similar avenues for inoculative wood frogs sylvatica)from southemOhio. Can.l. Zool. 65: 2062-2065. freezing exist in E. solidaginis larvae, such as the mouth and 1989.Seasonal variation in freezetolerance and ice contentof spiracles,but it was not determined in this study if their cuticle the treefrogHyla versicolor.J. Exp. Zool.249: 133-137. permits inoculative freezing. Lrn, R. E., Jn., andLrwrs, E. A. 1985.Effect of temperatureand Our study documentsthat external ice can causeinoculative durationofexposure on tissueice formationin thegali fly, Euosta freezingof supercooledR. sylvatica andE. sotidaginrsat high solidaginis(Diptera, Tephritidae). Cryo Lett. 6:25-34. subzerotemperatures that normaily iue not consideredcondu- LorsH,r.w,D.L. 1977.Temperature adaptation and effects of thermal cive to freezing. Moreover, limited evidenceis offered that this acclimationin Rana sylvatica and Rana catesbeiano.Cornp. -294. likely occurs within the hibernaculum of both species,but in Biochem.Physiol. 56A: 287 MecA,ntnun, physiological gall fly larvae, this effect is highly dependenton seasonaldrying D. L., and DeNoy, J. W. T. 1982. aspectsof overwintering in the boreal chorus (pseudacris of their host plants. Finally, the integument provides an frog triseriatamaculata). Comp. Biochem. physiol. 72A: adequateroute for inoculation in at least the wood frog. l4l-14j. Mon-nrssev,R. E., and Beusr, J. G. 1976.The ontogenyof cold physiol. Acknowledgements tolerancein thegall fly, Eurostasolidaginis. J. Insect 22: 431-437. We thankJamesIngold of ShippensburgUniversity and Scott Ror,c,s,R. R., Lre, R. E., Jn.,and Bnusr, J. G. 1986.Relationship of Moody of Ohio University for their assistancein collecting environmentalwater content to glycerolaccumulation in thefreeiing frogs during the courseof this srudy. A. L. DeVries (Univenity tolerantlarvae of Euostasolidaginis (Fitch). Cryo Len. 7:234- of lllinois) generouslyprovided the design for constructionof 245. the Ussing chamber.We thank J. S. Bale, T. N. Hansen,and Serr, R. W. 1950.Time as a factorin the freezingof undercooled insects. J. G. Baust for an advance copy of theix manuscript. J. p. Can.J. Res.Sect. D, 2E:285-291. Serr, R. W. 1957.Natural occurrence of glycerol Costanzoprovided commentson a draft of this manuscript.This in insectsand its relation to their ability to work was supported part survive freezing.Can. Entomol.E9: in by National Institutes of Health 49t-494. gant No. SAT I Rl5 HL40535-01 and National Science 1963.Delayed innoculative freezing ofinsects. Can. Entomol. Foundationgant No. DCB-8811317 (R.E.L.) and an Ohio 95: 1190-1202. UniversityResearch Committee gant (J.R.L.). Scnurp, W. D. 1982.Survival of frogsin low temperature.Science (Washington,D.C.), 215: 697-698. 1986.Winter ecology. Ekologiya, 6: 29-35. B,lu, J. S., H,lxseN,T. N., andBlusr, J. G. 1989.Nucleators and Snnteol, K., andRrrHrM,LA, A. 1988.Cold acclimation, inoculative sitesof nucleationin thefreeze tolerant larvae of thegallfly Eurosta freezing and slow cooling: essentialfactors contributing to t^he solidaginis(Fitch). J. InsectPhysiol. 35:291-298. freezing+olerancein diapausing larvae of Clrymomyzacostata Beusr, J. G. 1986. Insectcold hardiness:freezing tolerance and (Diptea:). Cryo Lett. 9: 5-10. avoidance-the Eurostamodel.1z Living in thecold. physiological Souran,L. 1978.Nucleating agents in thehemolymph of third instar and biochemicaladaptations. Edited by H. C. Heller, X. J. larvaeof Euostasolidaginis (Fitch) (Dipt., Tephritidae).Norw. J. Musacchia,andL. C. H. Wang.Elsevier, New York. pp. 125-130. Entomol.25: 187-188. Blusr, J. G., andLer, R. 8., Jn. 1981.Divergent mechanisms of Sronrv, K. B. 1984.Freeze tolerance in the frog, Rarn sylvatica. frost-hardinessin two populationsof thegall fly, Eurostasolidagin- Experientia,40:. 126l - 1262. sis.J. InsectPhysiol. 27:485-490. 1985. Freezetolerance in tenestrialfrogs. Cryo Lrtt. 6: 1982.Environmental triggers to cryoprotectantmodulation in I l5- 134. separatepopulations of the gall fly, Eurostasolidaginis. J. Insect Sronev, K. B., and Sronry, J. M. 1986. Freezetolerance and Physiol.2E:431-436. intoleranceas sffategies of winter survivalin tenestriallyhiircrnating Bnusr,J. G., andRores, R. R. 1985.Review-insect cold hardiness: amphibians.Comp. Biochem. Physiol. E3A: 613-6 17. factsand fancy. J. InsectPhysiol. 3l:755-759. 1987.Evaluation of the persistenceof freezetolerance in Blusr, J. G., GncNoee,R., Coxoox, G.,andMonrussny. R. E. tenesriallyhibernating frogs after spring emergence. Copeia, 1987: 1979.The divenity of overwinteringstrategies utilized by separate 720-726. populationsof gall insects.Physiol. Zool. 52: 572-580. 1988.Freeze tolerance in animals.Physiol. Rev. 68: 27-84. DurraeN,J. G. 1983.Insect antifreezesand ice-nucleatingagents. Tunxrn, J. D., ScHnc,c,J. D., andDeVntrs, A. L. 1985.Ocula Cryobiology,19: 613-627. freezingavoidance in antarcticfish. J. Exp. Biol. 118: l2l-131. DuuaN,J. G., Nrvex, L. G., Beers,J. M., Orsox,K. R., and UHrne, L. D. 1951.Biology and ecologyof the goldenrodgall fly, C.rsrnruNo, F. J. 1985.Freeze-tolerance adaptations, including Euostasolidagrnis (Fitch). CornellUniv. Agric. Exp. Stn. Mem. haemolymphprotein and lipoprotein nucleators, in thelarvae of the 300:l-51. craneflyTipula tivittata. J. InsectPhysiol. 3l: l-8 Zlcs.lru,rstx, K. E. 1985.Physiology of cold tolerancein insects. HErueN, S. I., andMrrrEn, D. A. 1971.In vitro techniquesfor Physiol.Rev. 65: 799-832.