Calcium signaling mediates cold sensingin insect tissuesNicholas M. Teetsa,1, Shu-Xia Yib, Richard E. Lee, Jr.b, and David L. Denlingera,c,1

aDepartment of Entomology, Ohio State University, Columbus, OH 43210; bDepartment of Zoology, Miami University, Oxford, OH 45056; and cDepartmentof Evolution, Ecology, and Organismal Biology, Ohio State University, Columbus, OH 43210

Contributed by David L. Denlinger, April 10, 2013 (sent for review January 30, 2013)

The ability to rapidly respond to changes in temperature is a criticaladaptation for insects and other ectotherms living in thermallyvariable environments. In a process called rapid cold hardening(RCH), insects significantly enhance cold tolerance following brief(i.e., minutes to hours) exposure to nonlethal chilling. Althoughthe ecological relevance of RCH is well-established, the underlyingphysiological mechanisms that trigger RCH are poorly understood.RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed bybrain-independent signaling mechanisms. We previously providedpreliminary evidence that calcium is involved in RCH, and here wefirmly establish that calcium signaling mediates cold sensing ininsect tissues. In tracheal cells of the freeze-tolerant goldenrod gallfly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase inintracellular calcium concentration as determined by live-cell con-focal imaging. Downstream of calcium entry, RCH conditions sig-nificantly increased the activity of calcium/calmodulin-dependentprotein kinase II (CaMKII) while reducing phosphorylation of theinhibitory Thr306 residue. Pharmacological inhibitors of calciumentry, calmodulin activation, and CaMKII activity all preventedex vivo RCH in midgut and salivary gland tissues, indicating thatcalcium signaling is required for RCH to occur. Similar results wereobtained for a freeze-intolerant species, adults of the flesh fly,Sarcophaga bullata, suggesting that calcium-mediated cold sens-ing is a general feature of insects. Our results imply that insecttissues use calcium signaling to instantly detect decreases in tem-perature and trigger downstream cold-hardening mechanisms.

calcium imaging | cold acclimation | environmental stress | overwintering |physiological ecology

Low temperature is one of the primary constraints for insectsand other ectotherms living in temperate and polar regions (1).

Although seasonal adaptations to cold stress, including envi-ronmentally programmed periods of dormancy called diapause,have been well-studied (2–5), physiological responses to suddenchanges in temperature have received less attention. In a processtermed rapid cold hardening (RCH), insects dramatically en-hance their cold tolerance in a matter of minutes to hours (6). Forexample, in the flesh fly, Sarcophaga crassipalpis, the first speciesin which RCH was described, exposure to 0 °C for as little as30 min significantly enhances cold tolerance at −10 °C (6). RCHhas since been described in dozens of insect species (7), includingboth freeze-intolerant (insects in which internal ice formation islethal) and freeze-tolerant species (insects that tolerate internalice formation) (8, 9). Naturally occurring thermoperiods canelicit RCH (10), and RCH preserves essential functions such ascourtship and mating (11, 12), supporting the relevance of thisprocess to natural populations.Although the ecological relevance of RCH has been estab-

lished, the physiological mechanisms are poorly understood.RCH results in a slight increase in the cryoprotectant glycerol (6,13), changes in cell membrane composition and fluidity (14–16),and up-regulation of a single heat shock protein in the brain (17).However, gene expression does not appear to be a major driverof RCH; in the flesh fly, Sarcophaga bullata, no transcripts (out

of ∼15,000 tested) were differentially expressed following 2 h ofRCH (18) whereas, in Drosophila melanogaster, RCH failed toincrease the expression of five genes previously linked to coldtolerance (19). Thus, we hypothesize that RCH is primarilymediated by second messenger systems that do not require thesynthesis of new gene products. Indeed, signaling pathways suchas MAP kinase (20, 21) and apoptosis signaling (22, 23) havebeen linked to RCH although the upstream cold-sensing mech-anisms that trigger these pathways are unknown.One of the most remarkable features of RCH is that isolated

tissues retain the capacity for RCH ex vivo (24, 25), indicatingthat cells directly respond to low temperature in the absence ofstimulation from the brain and hormones. However, the mech-anism by which tissues detect changes in temperature and triggerdownstream cold-hardening pathways is unknown. Chilling causesa rapid loss of ion homeostasis in insect tissues (26–33), but whetherthese ion movements play a role in tissue-level cold sensing has notbeen assessed. Cold acclimation in plants, which requires days toweeks for induction (34), is regulated by cold-induced calciuminflux (35–37). In addition, fruit flies (D. melanogaster) with amutant copy of the membrane protein dystroglycan have ele-vated levels of intracellular calcium, which correlates withimproved performance and survival at low temperatures (38).We recently provided pharmacological evidence that calciumand calmodulin are required for RCH in the Antarctic midge,Belgica antarctica (25), but cold-induced calcium signaling hasnot been examined in detail. Here, we provide evidence thatchilling rapidly elevates intracellular calcium, and that calcium-and calmodulin-dependent signaling events are required for coldsensing and RCH in insect tissues. Experiments were conductedin two well-studied models of insect cold tolerance, the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, and the freeze-intolerant flesh fly, S. bullata. RCH is the fastest cold-hardeningprocess that has been described, and we provide clear evidenceof the underlying cold-sensing mechanisms.

Results and DiscussionEx Vivo RCH in E. solidaginis. The goldenrod gall fly, E. solidaginis,is one of the most cold-hardy temperate species known and haslong been a model for studying freeze tolerance (39). Larvae ofE. solidaginis are capable of RCH (40) although the capacity forRCH in isolated tissues has not been addressed in this species.When midgut and salivary gland tissue from winter-acclimatizedthird instar larvae were removed and directly frozen at −20 °C,high mortality resulted. Cell survival of midgut tissue dropped to

Author contributions: N.M.T., R.E.L., and D.L.D. designed research; N.M.T. and S.-X.Y.performed research; N.M.T. and S.-X.Y. analyzed data; and N.M.T., S.-X.Y., R.E.L., andD.L.D. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. KC810053–KC810056).1Towhom correspondencemay be addressed. E-mail: teets.23@osu.edu or denlinger.1@osu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1306705110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306705110 PNAS Early Edition | 1 of 6

PHYS

IOLO

GY

∼50% (Fig. 1A) whereas that of the salivary gland was ∼60%(Fig. 1B), in contrast to untreated controls held at 4 °C, whichhad >90% survival (Fig. 1 A and B). However, when tissueswere first given a 1-h RCH period before freezing, in which tem-perature was gradually lowered from 4 °C to −20 °C, survivalincreased by 25–30% in each tissue (Fig. 1 A and B). This cold-hardening response occurred ex vivo in the absence of stimulationfrom nerves or hormones, indicating that RCH in tissues ofE. solidaginis is independent of the nervous system.

Chilling Increases Intracellular Calcium in Tissues of E. solidaginis.Using live-cell confocal imaging, we observed a 40% increase inintracellular calcium in tracheal cells of E. solidaginis when tem-perature was lowered from 25 °C to 0 °C at 1 °C/min (Fig. 1 C andD). Lowering temperature from 25 °C to 20 °C raised intracellularcalcium by 7%, and calcium rose continuously as temperaturedecreased. Maintaining temperature at 25 °C for the duration ofthe 40-min experiment failed to elicit a significant increase in cy-tosolic calcium (Fig. 1E). Cold-induced increases in cytosoliccalcium have also been demonstrated in plants (35) although coldexposure in plants causes a single, sharp spike in intracellularcalcium, suggesting that a different mechanism is at play. Modestelevation of intracellular calcium at low temperature is also knownin fish (41) and mammals (42, 43) although not in the context ofcold hardening. In insects, disruption of ion gradients duringchilling is responsible for the loss of neuromuscular function at lowtemperature (26–33), but whether these ion movements are usedto trigger cold hardening has not been addressed.

RCH Activates Calcium-Dependent Signaling Pathways. Downstreamof calcium entry into the cell, chilling activated calcium-dependentsignaling pathways in E. solidaginis. Specifically, we monitored theactivity and phosphorylation state of the calcium-dependent sig-naling enzyme calcium/calmodulin dependent protein kinase II(CaMKII) in response to low temperature. CaMKII is a multi-functional signaling enzyme that regulates numerous metabolicand gene expression processes (44), and whereas this enzyme hasbeen linked to cellular stress in mammalian systems (e.g., ref. 45),it has not been associated with environmental stress. In larvae ofE. solidaginis, RCH conditions induced a significant increase (from0.65 to 0.88 pmol·min−1·μg protein−1) in the activity of CaMKII(Fig. 1F). Although this protein was activated during RCH, afterprolonged freezing at −15 °C for 24 h, with and without 2 h re-covery at 18 °C, CaMKII activity decreased to levels that wereindistinguishable from both control and RCH-treated larvae (Fig.1F). Thus, activation of this protein occurred specifically duringthe RCH period. Additionally, RCH decreased phosphorylationat the inhibitory Thr306 residue by 60% (Fig. 1G andH), which isconsistent with enzyme activation (44). In contrast, chilling at 4 °Cfor 24 h and 2 h recovery from freezing significantly increased thephosphorylation ratio (Fig. 1G and H), suggesting deactivation ofthis enzyme in response to prolonged chilling and recovery fromfreezing. We cloned and sequenced full-length calmodulin (Gen-Bank accession no. KC810053) and CaMKII (GenBank accessionno. KC810054) transcripts from E. solidaginis and measured theirtissue-specific expression. Predicted amino acid sequences forboth proteins indicated that this signaling axis is highly conservedacross insects and mammals (Figs. S1A and S2A), and transcriptswere expressed in all six larval tissues tested (Figs. S1B and S2B),suggesting that these genes could mediate cold sensing throughoutthe body.

Fig. 1. Rapid cold hardening reduces freezing injury in tissues of thegoldenrod gall fly, E. solidaginis and activates calcium-signaling pathways.(A and B) Effect of ex vivo RCH (slow ramping from 4 °C to −20 °C over 1 h)on cell viability of (A) midgut and (B) salivary gland following freezing at−20 °C. (C–E) Intracellular calcium as a function of temperature in trachealcells of E. solidaginis. (F) Activity of calcium/calmodulin-dependent proteinkinase II (CaMKII) following RCH, freezing for 24 h, and freezing with 2 hrecovery. (G) Levels of both total CaMKII and phospho-Thr306 CaMKII inresponse to low temperature. (H) The ratio of phospho-Thr306 CaMKII tototal CaMKII in response to low temperature. In A and B, “Control” tissueswere held at 4 °C for 3 h, “Directly Frozen” tissues were directly transferredfrom 18 °C to −20 °C for 2 h whereas “RCH” tissues were gradually chilledfrom 4 °C to −20 °C over 1 h, then held at −20 °C for 2 h. In C, relative calciumconcentration of a representative tracheal end cell is indicated by the pro-vided color scale. In D and E, an asterisk indicates a significant differencebetween a particular time point and the initial calcium concentration (re-peated measures ANOVA, post hoc Bonferroni, P < 0.05). In F–H, CaMKII

activity and phosphorylation were measured in whole-body protein extracts.All values are mean ± SEM, n ≥ 4, with different letters indicating significantdifferences (ANOVA, Tukey, P < 0.05).

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1306705110 Teets et al.

Functional Significance of Calcium Signaling During RCH. To de-termine the functional significance of calcium signaling during coldsensing, we used pharmacological inhibitors to block various com-ponents of calcium-signaling pathways during RCH. In midgut andsalivary gland tissues of larval E. solidaginis, removing calcium fromthe medium, blocking calcium channels with LaCl3, and chelatingintracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, acetoxymethyl ester (BAPTA-AM) all inhibitedex vivoRCH, reducing survival 17–47% relative to tissues incubatedwith calcium (Fig. 2). In addition, blocking calmodulin with the drugN-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochlo-ride (W-7) and inhibiting CaMKII with 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-meth-ylbenzylamine (KN-93) similarly reduced cell survival followingRCH (Fig. 2). However, inhibition of inositol triphosphate (IP3)-mediated calcium signaling had no effect on survival, suggestingthat IP3-mediated calcium release is not involved in cold sensing.These drugs were not toxic to tissues held at 4 °C and had littleeffect on the survival of tissues directly frozen at −20 °C (Fig. S3Aand B). Furthermore, at milder temperatures, inhibition of calciumsignaling reduced baseline freezing tolerance. After direct freez-ing at −17.5 °C, cell survival was significantly higher than at−20 °C(71.1% vs. 51.1% formidgut, 85.9% vs. 60.5% for salivary gland; Fig.S3 C and D), but inhibition of calcium signaling reduced survivalat −17.5 °C (Fig. S3 E and F). These pharmacological datademonstrate that calcium entry is not simply a symptom of lowtemperature but is an essential cold-sensing mechanism medi-ating rapid responses to cold.

Calcium Signaling also Mediates RCH in a Freeze-Intolerant Insect.The above results clearly demonstrate a role for calcium signal-ing during RCH in larvae of E. solidaginis, a freeze-tolerant or-ganism. Subsequent experiments indicated that calcium signalingalso governs cold sensing and RCH in a freeze-intolerant species,adults of the flesh fly S. bullata. Salivary gland tissues from thisspecies were more amenable to calcium imaging, allowing a morein-depth analysis of the dynamics of cold-induced calcium move-ments. Chilling to 0 °C elicited a 70% increase in intracellularcalcium in adult salivary gland tissue (Fig. 3 A and B) whereasmaintaining tissues at 25 °C for the duration of the experimentfailed to evoke a calcium response (Fig. 3C). When the coolingrate was increased to 2 °C/min, calcium concentrations showeda similar increase although the concentration at a given temper-ature was consistently lower than when tissues were cooled at 1 °C/min (Fig. 3D). Warming back to 25 °C from 0 °C caused an im-mediate reversal in calcium flux (Fig. 3E), which may in part ex-plain the rapid attenuation of RCH upon warming (46).Like E. solidaginis, RCH in isolated tissues of S. bullata

was dependent on calcium-signaling pathways. For these experi-ments, RCH was generated by directly transferring tissues to 0 °Cfor 2 h, before transferring tissues to the discriminating temper-ature (−14 °C) for 2 h. This stepwise protocol is the typical ap-proach for demonstrating RCH in the laboratory (7). In bothmidgut and fat body tissue from adults of S. bullata, blockingcalcium entry and calmodulin activation prevented RCH (Fig. 3F–H) whereas these drugs had no effect on control tissues andtissues directly cold shocked at −14 °C (Fig. S3 G and H). In thisspecies, calmodulin (GenBank accession no. KC810055) andCaMKII (GenBank accession no. KC810056) were expressed inall postembryonic developmental stages and in nearly every tissue(Figs. S4 and S5), suggesting that this signaling axis could mediatecold sensing throughout the body in every developmental stage.Thus, although the capacity for freeze tolerance has evolvednumerous times in the insect lineage (47), cold-induced calciumsignaling appears to be a general response to chilling in insects.

Conceptual Model for Calcium-Mediated Cold Sensing. The resultspresented here represent a significant advance in our understanding

of the cell physiology of insect low temperature response. A hy-pothetical model for the mode of calcium entry and the down-stream targets of calcium signaling in response to low temperatureis presented in Fig. 4. Low temperature directly inhibits ATP-de-pendent calcium-exporting functions, such as the Na+/Ca2+-exchanger coupled to Na+/K+-ATPase, causing a gradual leak ofcalcium ions into the cell (33). This mechanism of cold-induced

Fig. 2. Pharmacological inhibition of calcium signaling blocks rapid coldhardening in tissues of E. solidaginis. (A) Fluorescent cell viability images ofmidgut tissue incubated with the indicated drug at the following temper-ature conditions: Control, 4 °C/3 h; Directly Frozen, direct transfer to −20 °Cfor 2 h; RCH, slow ramp from 4 °C to −20 °C over 1 h, then held at −20 °C for2 h. Green nuclei represent live cells, and red nuclei represent dead cells.(B and C) Quantified viability results for (B) midgut and (C) salivary glandtissue. All values are mean ± SEM, n = 4, and different letters indicate sig-nificant differences among the three temperature treatments where anasterisk indicates a significant reduction in survival in the RCH group relativeto tissues incubated with calcium present (ANOVA, Tukey, P < 0.05). NCF,nominally calcium free medium. LaCl3 is a general calcium channel blocker,BAPTA is an intracellular calcium chelator, W-7 is an antagonist of the cal-cium binding protein calmodulin, and KN-93 inhibits activation of CaMKIIwhereas 2-APB is an inhibitor of IP3-mediated calcium signaling.

Teets et al. PNAS Early Edition | 3 of 6

PHYS

IOLO

GY

calcium influx is different from cold sensing in the nervous system,which involves direct activation of excitable, cold-sensitive transientreceptor potential channels in sensory neurons (48) that carry thesignal to specific regions of the brain (49). Although we cannot ruleout that RCH is simply responding to cold injury, with calciuminflux as the token symptom, the observation that calcium levelsclosely track environmental temperature (Fig. 1 C and D and 3 B–E) suggests a general cold-sensing role for calcium. Although thedownstream targets of calcium signaling during RCH have notbeen established, calcium signaling interacts with a number ofprocesses known to be important for cold hardening, includingapoptosis signaling (22, 23) and carbohydrate mobilization (50). Inthe cold hardy atsugari mutant of D. melanogaster, increased in-tracellular calcium improves cold tolerance by boosting mito-chondrial metabolism at low temperature (38), and subsequentexperiments will test whether metabolic changes associated withRCH (e.g., 13, 18, 51) are indeed calcium-regulated. Also, al-though the present study addressed only the calcium/calmodulin/CaMKII signaling axis, several other calcium-dependent signalingpathways are known to mediate stress responses. For example, the

calcium-dependent enzyme apoptosis signal-regulating kinase Iregulates p38 MAP kinase signaling (52), thus providing a poten-tial link between calcium and p38 signaling (20) during RCH.Calcium signaling provides a direct, intuitive means by which

isolated insect tissues detect and respond to low temperature.Although the sensing mechanisms governing behavioral responsesto low temperature in the nervous system are well-established(48), cold-sensing mechanisms in nonnervous tissues have beenhitherto unexplored. Understanding the mechanisms by whichinsects tolerate low temperature is essential for predicting theirresponse to a changing climate (53). Furthermore, mechanismsof cold hardening can be exploited to manipulate populations ofinsect pests (54). Our group has recently explored the possibilityof disrupting overwintering diapause to control pest populations(55), and we envision disruption of acute cold hardening to be anequally effective strategy.

MethodsAnimals. Galls containing third instar larvae of E. solidaginis, located on thestems of senesced Solidago canidensis plants, were collected from various

Fig. 3. Calcium signaling also mediates cold sensing and rapid cold hardening in a freeze-intolerant fly, S. bullata. Intracellular calcium concentration insalivary glands in response to (A and B) chilling at 1 °C/min, (C) control conditions of 25 °C, (D) chilling at 2 °C/min, and (E) chilling at 1 °C/min followed bywarming at 1 °C/min. (F) Fluorescent cell viability images of midgut tissue incubated with the indicated drug at the following temperature conditions: Control:25 °C/4 h; Cold Shock: −14 °C/2 h; RCH: 0 °C/2 h, −14 °C/2 h. Green nuclei represent live cells and red nuclei represent dead cells. (G and H) Quantified viabilityresults for (G) midgut and (H) fat body tissue. Values are mean ± SEM, n = 3 for (B–D), n = 2 for (E), n = 4 for (G and H). In A, relative calcium concentration ofa representative segment of salivary gland tissue is indicated by the provided color scale. In B–E, an asterisk indicates a significant difference between aparticular time point and the initial calcium concentration (repeated measures ANOVA, post hoc Bonferroni, P < 0.05). In G and H, an asterisk indicatesa significant reduction in survival in the RCH group relative to tissues incubated with calcium present (ANOVA, Tukey, P < 0.05). NCF, nominally calciumfree medium. LaCl3 is a general calcium channel blocker, BAPTA is an intracellular calcium chelator, W-7 is an antagonist of the calcium binding proteincalmodulin, and KN-93 inhibits activation of CaMKII whereas 2-APB is an inhibitor of IP3-mediated calcium signaling.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1306705110 Teets et al.

goldenrod fields in central and southwest Ohio from September to January2007–2012. Galls were stored at 18 °C, and larvae were removed just beforeexperimentation. Flesh flies, S. bullata, were laboratory-reared according toDenlinger et al. (56) under nondiapausing conditions (25 °C, 16:8 light:dark).Adults were fed sugar and liver ad libitum, and males were used for ex-periments 4–8 d after eclosion.

Calcium Imaging. Tissues from E. solidaginis and S. bullata were dissected inCoast’s solution (57) containing (in mM) 100 NaCl, 8.6 KCl, 4.0 NaHCO3, 4.0NaH2PO4-H2O, 1.5 CaCl2-2H2O, 8.5 MgCl2-6H2O, 24 glucose, 25 Hepes, and56 sucrose. For experiments with E. solidaginis, the Coast’s solution wassupplemented with 250 mM glycerol. After dissection, tissues were loadedwith 10 μM fluo-3 AM (Life Technologies) for 1 h at room temperature. Theloading solution contained 0.2% pluronic F-127 (Life Technologies) to dis-perse the dye. After loading, cells were rinsed twice in Coast’s solution, keptin the dark at room temperature, and imaged within the next 3 h. Pre-liminary experiments established that tracheal cells from E. solidaginis andsalivary gland cells from S. bullata were most amenable to calcium imaging.Other tissues were difficult to maintain in the proper focal plane or failedto take up the calcium imaging dye. Tissues were imaged with a VisitechInfinity3 Hawk 2D Array live-cell confocal microscope at the Ohio StateCampus Microscopy and Imaging Facility. Cells were excited at 488 nm, andfluorescence at 520 nM was measured every 10 s for the duration of theexperiment. Temperature was controlled with an Instec TSA02i invertedmicroscope thermal stage (Instec). Although images were acquired every10 s, low temperature caused a slight drift in the focal plane in most sam-ples, necessitating periodic refocusing. As a result, samples were refocusedevery 2.5–5 min, so results are presented only in 2.5- to 5-min increments.

Fluorescence was recorded in a region of interest that consisted of a singletracheal end cell in E. solidaginis and four to five salivary gland epithelialcells in S. bullata. Intracellular calcium concentration was calculated accordingthe following equation: [Ca2+]i = Kd(F/Fo)/(Kd/[Ca

2+]i-rest + 1 − F/Fo), where F isthe fluorescence, Fo is the initial fluorescence, and [Ca2+]i-rest is the restingcalcium concentration, which was estimated to be 100 nm, based on mea-surements of blow fly salivary glands conducted by Zimmermann and Walz(58). The Kd for fluo-3 was corrected for changes in temperature accordingto Woodruff et al. (59).

CaMKII Assays. CaMKII activity was measured with the Signatect CaMKII AssayKit (Promega). In this assay, CaMKII transfers phosphate from [γ-32P]ATPto a synthetic substrate for CaMKII, which is immobilized on a biotinylatedmembrane and measured with a scintillation counter. Protein was extractedfrom whole larvae of E. solidaginis with radioimmunoprecipitation assaybuffer containing the Halt Protease and Phosphatase Inhibitor mixture(ThermoFisher Scientific), and 25 μg of protein was loaded into each re-action. For Western blotting, protein samples were generated in the samemanner, and 35 μg of protein from each sample was loaded into a 4–15%gradient SDS/PAGE gel. Western blotting was conducted according to Yiet al. (23). Rabbit anti-CaMKII (total) was obtained from Santa Cruz Bio-technology and rabbit anti-CAMKII (pThr305) was obtained from Milliporewhereas mouse anti-β-tubulin was obtained from the Developmental Stud-ies Hybridoma Bank. Densitometry was conducted with AlphaView SAsoftware (ProteinSimple), with each sample normalized to β-tubulin. EachWestern blot was repeated with six independent biological replicates. Toverify whether mammalian-based assays and antibodies would work, wecloned and characterized full-length calmodulin and CaMKII cDNA se-quences using the Clontech SMARTer RACE kit (Clontech Laboratories).Additionally, in E. solidaginis, we measured tissue specific expression inlarval brains, midguts, fat body, salivary glands, Malpighian tubules, andepidermis using RT-PCR (Figs. S1 and S2). In S. bullata, we measured bothdevelopmental and tissue-specific expression profiles (Figs. S4 and S5).Primer sequences for RACE and PCR detection of transcripts are providedin Table S1.

Cell Viability Assays. Tissues were dissected in Coast’s solution at roomtemperature. Tissues from E. solidaginis were exposed to the followingtemperature conditions: control (4 °C, 3 h), directly frozen (directly trans-ferred from room temperature to −17.5 or −20 °C, and held there for 2 h),and RCH (slowly ramped from 4 °C to test temperature over 1 h, then heldat test temperature for 2 h). For S. bullata, tissues were exposed to control(25 °C, 4 h), cold shock (−14 °C, 2 h), and RCH (0 °C 2h, −14 °C 2h) conditions;all tissues remained supercooled at −14 °C. Cell viability was assessed with theLIVE/DEAD sperm viability assay (Life Technologies) (24), which is a mem-brane integrity assay consisting of SYBR green and propidium iodide. In thisassay, living cells with intact membranes fluoresce green whereas dead cellswith damaged membranes fluoresce red. Viability is expressed as the per-centage survival based on the counts of 300 cells per sample. To test whethercalcium signaling is essential for RCH, tissues were exposed to the followingsolutions and drugs to manipulate calcium signaling: nominally calcium free(NCF) medium, Coast’s solution prepared without calcium; 250 μM LaCl3,a general calcium channel blocker; 100 μM BAPTA-AM, an intracellular cal-cium chelator; 50 μM W-7, an inhibitor of calmodulin; 100 μM KN-93, aninhibitor of CaMKII, and 10 μM 2-aminoethoxydiphenyl borate (2-APB), aninhibitor of IP3-mediated calcium signaling. Tissues were loaded with drugsfor 30–60 min before conducting the same temperature experimentsdescribed above.

Statistics. All data are expressed as mean ± SE. Data from calcium-imagingexperiments were analyzed with repeated measures ANOVA and a post hocBonferroni test. All other means were compared with ANOVA and Tukey’spost hoc multiple comparisons procedure in JMP9 (SAS Institute). For calcium-imaging experiments, n = 2–5 for each experiment whereas, for CaMKII assays,n = 5–6 for each treatment. Cell viability assays were conducted with fourbiological replicates per treatment, and data were arcsin square-root trans-formed before analysis.

ACKNOWLEDGMENTS. We thank Sarah Cole, Brian Kemmenoe, RichardMontione, and other members of the Ohio State Campus Microscopy andImaging Facility for assistance with these experiments. We also thankDr. Michael Ibba for providing laboratory space for experiments requiringradiolabeled substrates, Josh Stapleton for help with densitometry, andmembers of the Laboratory for Ecophysiological Cryobiology at MiamiUniversity for assistance with gall collecting. Peter Piermarini and Larry

Fig. 4. Working model for the role of calcium signaling during cold sensingand rapid cold hardening. Low temperature causes an increase in intracellularcalcium concentration, and we hypothesize that this calcium influx occurs bylow temperature inhibition of ATP-dependent calcium export mechanismscoupled with calcium entry through calcium leak channels (CLC). In thismodel, low temperature inhibits the activity of both sarcoplasmic endo-plasmic reticulum calcium ATPase (SERCA) and sodium/potassium ATPase(Na/K ATPase) coupled to the sodium calcium exchanger (NCX). Inside the cell,calcium, via calcium/calmodulin-dependent protein kinase II (CaMKII) andother unknown mechanisms, triggers pathways involved in rapid cold hard-ening, thereby enhancing the cell’s cold tolerance. Dashed lines and arrowsindicate speculative relationships that were not experimentally determinedin the present study.

Teets et al. PNAS Early Edition | 5 of 6

PHYS

IOLO

GY

Phelan, Ohio State University, kindly provided comments on a draft of thismanuscript. We acknowledge Brent Sinclair, University of Western Ontario,

and John Duman, University of Notre Dame, for critically reviewing the paper.This work was supported by National Science Foundation Grant IOS-0840772.

1. Denlinger DL, Lee RE (1998) Physiology of cold sensitivity. Temperature Sensitivity inInsects and Application in Integrated Pest Management, eds Hallman GJ, DenlingerDL (Westview, Boulder, CO), pp 55–95.

2. Denlinger DL, Yocum GD, Rinehart JP (2005) Hormonal control of diapause. Com-prehensive Insect Molecular Science, eds Gilbert LI, Iatrou K, Gill S (Elsevier, Am-sterdam), pp 615–650.

3. Denlinger DL (1991) Relationship between cold hardiness and diapause. Insectsat Low Temperature, eds Lee RE, Denlinger DL (Chapman and Hall, New York),pp 174–198.

4. Hahn DA, Denlinger DL (2011) Energetics of insect diapause. Annu Rev Entomol56:103–121.

5. Kostál V (2006) Eco-physiological phases of insect diapause. J Insect Physiol 52(2):113–127.

6. Lee RE, Jr., Chen CP, Denlinger DL (1987) A rapid cold-hardening process in insects.Science 238(4832):1415–1417.

7. Lee RE, Denlinger DL (2010) Rapid cold-hardening: Ecological significance and un-derpinning mechanisms. Low Temperature Biology of Insects, eds Denlinger DL, LeeRE (Cambridge Univ Press, Cambridge, UK), pp 35–58.

8. Lee RE, Jr., et al. (2006) Rapid cold-hardening increases the freezing tolerance of theAntarctic midge Belgica antarctica. J Exp Biol 209(3):399–406.

9. Everatt MJ, Worland MR, Bale JS, Convey P, Hayward SAL (2012) Pre-adapted tothe maritime Antarctic?—Rapid cold hardening of the midge, Eretmoptera murphyi.J Insect Physiol 58(8):1104–1111.

10. Kelty J (2007) Rapid cold-hardening of Drosophila melanogaster in a field setting.Physiol Entomol 32(4):343–350.

11. Shreve SM, Kelty JD, Lee RE, Jr. (2004) Preservation of reproductive behaviors duringmodest cooling: Rapid cold-hardening fine-tunes organismal response. J Exp Biol207(11):1797–1802.

12. Rinehart JP, Yocum GD, Denlinger DL (2000) Thermotolerance and rapid cold hard-ening ameliorate the negative effects of brief exposures to high or low temperatureson fecundity in the flesh fly, Sarcophaga crassipalpis. Physiol Entomol 25(4):330–336.

13. Michaud MR, Denlinger DL (2007) Shifts in the carbohydrate, polyol, and amino acidpools during rapid cold-hardening and diapause-associated cold-hardening in fleshflies (Sarcophaga crassipalpis): A metabolomic comparison. J Comp Physiol B 177(7):753–763.

14. Lee RE, Jr., Damodaran K, Yi S-X, Lorigan GA (2006) Rapid cold-hardening increasesmembrane fluidity and cold tolerance of insect cells. Cryobiology 52(3):459–463.

15. Michaud MR, Denlinger DL (2006) Oleic acid is elevated in cell membranes duringrapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis.J Insect Physiol 52(10):1073–1082.

16. Overgaard J, Sørensen JG, Petersen SO, Loeschcke V, Holmstrup M (2005) Changes inmembrane lipid composition following rapid cold hardening in Drosophila melanogaster.J Insect Physiol 51(11):1173–1182.

17. Li A, Denlinger DL (2008) Rapid cold hardening elicits changes in brain protein pro-files of the flesh fly, Sarcophaga crassipalpis. Insect Mol Biol 17(5):565–572.

18. Teets NM, et al. (2012) Combined transcriptomic and metabolomic approach uncoversmolecular mechanisms of cold tolerance in a temperate flesh fly. Physiol Genomics44(15):764–777.

19. Sinclair BJ, Gibbs AG, Roberts SP (2007) Gene transcription during exposure to, andrecovery from, cold and desiccation stress in Drosophila melanogaster. Insect Mol Biol16(4):435–443.

20. Fujiwara Y, Denlinger DL (2007) p38 MAPK is a likely component of the signaltransduction pathway triggering rapid cold hardening in the flesh fly Sarcophagacrassipalpis. J Exp Biol 210(18):3295–3300.

21. Li F-F, Xia J, Li J-M, Liu S-S, Wang X-W (2012) p38 MAPK is a component of the signaltransduction pathway triggering cold stress response in the MED cryptic species ofBemisia tabaci. J Integr Agric 11(2):303–311.

22. Yi S-X, Lee RE, Jr. (2011) Rapid cold-hardening blocks cold-induced apoptosis byinhibiting the activation of pro-caspases in the flesh fly Sarcophaga crassipalpis.Apoptosis 16(3):249–255.

23. Yi S-X, Moore CW, Lee RE, Jr. (2007) Rapid cold-hardening protects Drosophilamelanogaster from cold-induced apoptosis. Apoptosis 12(7):1183–1193.

24. Yi S-X, Lee RE, Jr. (2004) In vivo and in vitro rapid cold-hardening protects cells fromcold-shock injury in the flesh fly. J Comp Physiol B 174(8):611–615.

25. Teets NM, et al. (2008) Rapid cold-hardening in larvae of the Antarctic midge Belgicaantarctica: Cellular cold-sensing and a role for calcium. Am J Physiol Regul IntegrComp Physiol 294(6):R1938–R1946.

26. Macmillan HA, Sinclair BJ (2011) Mechanisms underlying insect chill-coma. J InsectPhysiol 57(1):12–20.

27. MacMillan HA, Sinclair BJ (2011) The role of the gut in insect chilling injury: Cold-induced disruption of osmoregulation in the fall field cricket, Gryllus pennsylvanicus.J Exp Biol 214(5):726–734.

28. MacMillan HA, Williams CM, Staples JF, Sinclair BJ (2012) Reestablishment of ionhomeostasis during chill-coma recovery in the cricket Gryllus pennsylvanicus. Proc NatlAcad Sci USA 109(50):20750–20755.

29. Kostál V, Renault D, Mehrabianová A, Bastl J (2007) Insect cold tolerance and repair ofchill-injury at fluctuating thermal regimes: Role of ion homeostasis. Comp BiochemPhysiol A Mol Integr Physiol 147(1):231–238.

30. Kostál V, Vambera J, Bastl J (2004) On the nature of pre-freeze mortality in insects:Water balance, ion homeostasis and energy charge in the adults of Pyrrhocorisapterus. J Exp Biol 207(9):1509–1521.

31. Kostál V, Yanagimoto M, Bastl J (2006) Chilling-injury and disturbance of ion ho-meostasis in the coxal muscle of the tropical cockroach (Nauphoeta cinerea). CompBiochem Physiol B Biochem Mol Biol 143(2):171–179.

32. Kristiansen E, Zachariassen KE (2001) Effect of freezing on the transmembranedistribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus(Diptera, Xylophagidae). J Insect Physiol 47(6):585–592.

33. Zachariassen KE, Kristiansen E, Pedersen SA (2004) Inorganic ions in cold-hardiness.Cryobiology 48(2):126–133.

34. Thomashow MF (1999) Plant cold acclimation: Freezing tolerance genes and regula-tory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599.

35. Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis in-volves two cellular pools and a change in calcium signature after acclimation. PlantCell 8(3):489–503.

36. Monroy AF, Dhindsa RS (1995) Low-temperature signal transduction: induction ofcold acclimation-specific genes of alfalfa by calcium at 25 °C. Plant Cell 7(3):321–331.

37. Monroy AF, Sarhan F, Dhindsa RS (1993) Cold-induced changes in freezing tolerance,protein-phosphorylation, and gene exression - Evidence for a role of calcium. PlantPhysiol 102(4):1227–1235.

38. Takeuchi K, et al. (2009) Changes in temperature preferences and energy homeostasisin dystroglycan mutants. Science 323(5922):1740–1743.

39. Bennett VA, Lee RE (1997) Modeling seasonal changes in intracellular freeze-toleranceof fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J Exp Biol200(1):185–192.

40. Levis NA, Yi SX, Lee RE, Jr. (2012) Mild desiccation rapidly increases freeze toleranceof the goldenrod gall fly, Eurosta solidaginis: Evidence for drought-induced rapidcold-hardening. J Exp Biol 215(21):3768–3773.

41. Shiels HA, Di Maio A, Thompson S, & Block BA (2011) Warm fish with cold hearts:Thermal plasticity of excitation-contraction coupling in bluefin tuna. Proc R Soc B BiolSci 278(1702):18–27.

42. Haddad P, Cabrillac JC, Piche D, Musallam L, Huet PM (1999) Changes in intracellularcalcium induced by acute hypothermia in parenchymal, endothelial, and Kupffer cellsof the rat liver. Cryobiology 39(1):69–79.

43. Wang SQ, Lakatta EG, Cheng H, Zhou ZQ (2002) Adaptive mechanisms of intracellularcalcium homeostasis in mammalian hibernators. J Exp Biol 205(19):2957–2962.

44. Colbran RJ (2004) Targeting of calcium/calmodulin-dependent protein kinase II.Biochem J 378(1):1–16.

45. Timmins JM, et al. (2009) Calcium/calmodulin-dependent protein kinase II linksER stress with Fas and mitochondrial apoptosis pathways. J Clin Invest 119(10):2925–2941.

46. Coulson SJ, Bale JS (1990) Characterization and limitations of the rapid cold-hard-ening response in the housefly Musca domestica (Diptera, Muscidae). J Insect Physiol36(3):207–211.

47. Sinclair BJ, Addo-Bediako A, Chown SL (2003) Climatic variability and the evolution ofinsect freeze tolerance. Biol Rev Camb Philos Soc 78(2):181–195.

48. Rosenzweig M, Kang KJ, Garrity PA (2008) Distinct TRP channels are required forwarm and cool avoidance in Drosophila melanogaster. Proc Natl Acad Sci USA 105(38):14668–14673.

49. Gallio M, Ofstad TA, Macpherson LJ, Wang JW, Zuker CS (2011) The coding of tem-perature in the Drosophila brain. Cell 144(4):614–624.

50. Johnson LN (1992) Glycogen phosphorylase: Control by phosphorylation and allostericeffectors. FASEB J 6(6):2274–2282.

51. Overgaard J, et al. (2007) Metabolomic profiling of rapid cold hardening and coldshock in Drosophila melanogaster. J Insect Physiol 53(12):1218–1232.

52. Takeda K, et al. (2004) Involvement of ASK1 in Ca2+-induced p38 MAP kinase acti-vation. EMBO Rep 5(2):161–166.

53. Bale JS, Hayward SAL (2010) Insect overwintering in a changing climate. J Exp Biol213(6):980–994.

54. Lee RE, Lee MR, Strong-Gunderson JM (1993) Insect cold-hardiness and ice nucleatingactive microorganisms including their potential use for biological-control. J InsectPhysiol 39(1):1–12.

55. Zhang Q, Nachman RJ, Kaczmarek K, Zabrocki J, Denlinger DL (2011) Disruption ofinsect diapause using agonists and an antagonist of diapause hormone. Proc NatlAcad Sci USA 108(41):16922–16926.

56. Denlinger DL (1972) Induction and termination of pupal diapause in Sarcophaga(Diptera - Sarcophagidae). Biol Bull 142(1):11–24.

57. Coast GM, Krasnoff SB (1988) Fluid secretion by single isolated Malpighian tubules ofthe house cricket, Acheta domesticus, and their response to diuretic hormone. PhysiolEntomol 13(4):381–391.

58. Zimmermann B, Walz B (1999) The mechanism mediating regenerative intercellularCa2+ waves in the blowfly salivary gland. EMBO J 18(12):3222–3231.

59. Woodruff ML, et al. (2002) Measurement of cytoplasmic calcium concentration in therods of wild-type and transducin knock-out mice. J Physiol 542(3):843–854.

6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1306705110 Teets et al.