Calcium Signaling Mediates Cold Sensing in Insect Tissues

Calcium signaling mediates cold sensing in insect tissues

Nicholas 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 cDepartment of 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 critical of ∼15,000 tested) were differentially expressed following 2 h of adaptation for insects and other ectotherms living in thermally RCH (18) whereas, in Drosophila melanogaster, RCH failed to variable environments. In a process called rapid cold hardening increase the expression of five genes previously linked to cold (RCH), insects significantly enhance cold tolerance following brief tolerance (19). Thus, we hypothesize that RCH is primarily (i.e., minutes to hours) exposure to nonlethal chilling. Although mediated by second messenger systems that do not require the the ecological relevance of RCH is well-established, the underlying synthesis of new gene products. Indeed, signaling pathways such physiological mechanisms that trigger RCH are poorly understood. as MAP kinase (20, 21) and apoptosis signaling (22, 23) have RCH can be elicited in isolated tissues ex vivo, suggesting cold- been linked to RCH although the upstream cold-sensing mech- sensing and downstream hardening pathways are governed by anisms that trigger these pathways are unknown. brain-independent signaling mechanisms. We previously provided One of the most remarkable features of RCH is that isolated preliminary evidence that calcium is involved in RCH, and here we tissues retain the capacity for RCH ex vivo (24, 25), indicating firmly establish that calcium signaling mediates cold sensing in that cells directly respond to low temperature in the absence of insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall stimulation from the brain and hormones. However, the mech- fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in anism by which tissues detect changes in temperature and trigger intracellular calcium concentration as determined by live-cell con- downstream cold-hardening pathways is unknown. Chilling causes focal imaging. Downstream of calcium entry, RCH conditions sig- a rapid loss of ion homeostasis in insect tissues (26–33), but whether PHYSIOLOGY nificantly increased the activity of calcium/calmodulin-dependent these ion movements play a role in tissue-level cold sensing has not protein kinase II (CaMKII) while reducing phosphorylation of the been assessed. Cold acclimation in plants, which requires days to inhibitory Thr306 residue. Pharmacological inhibitors of calcium weeks for induction (34), is regulated by cold-induced calcium entry, calmodulin activation, and CaMKII activity all prevented influx (35–37). In addition, fruit flies (D. melanogaster)witha ex vivo RCH in midgut and salivary gland tissues, indicating that mutant copy of the membrane protein dystroglycan have ele- calcium signaling is required for RCH to occur. Similar results were vated levels of intracellular calcium, which correlates with obtained for a freeze-intolerant species, adults of the flesh fly, improved performance and survival at low temperatures (38). Sarcophaga bullata, suggesting that calcium-mediated cold sens- We recently provided pharmacological evidence that calcium ing is a general feature of insects. Our results imply that insect and calmodulin are required for RCH in the Antarctic midge, tissues use calcium signaling to instantly detect decreases in tem- Belgica antarctica (25), but cold-induced calcium signaling has perature and trigger downstream cold-hardening mechanisms. not been examined in detail. Here, we provide evidence that chilling rapidly elevates intracellular calcium, and that calcium- calcium imaging | cold acclimation | environmental stress | overwintering | and calmodulin-dependent signaling events are required for cold physiological ecology sensing and RCH in insect tissues. Experiments were conducted in two well-studied models of insect cold tolerance, the freeze- ow temperature is one of the primary constraints for insects tolerant goldenrod gall fly, Eurosta solidaginis, and the freeze- Land other ectotherms living in temperate and polar regions (1). intolerant flesh fly, S. bullata. RCH is the fastest cold-hardening Although seasonal adaptations to cold stress, including envi- process that has been described, and we provide clear evidence ronmentally programmed periods of dormancy called diapause, of the underlying cold-sensing mechanisms. have been well-studied (2–5), physiological responses to sudden changes in temperature have received less attention. In a process Results and Discussion termed rapid cold hardening (RCH), insects dramatically en- Ex Vivo RCH in E. solidaginis. The goldenrod gall fly, E. solidaginis, hance their cold tolerance in a matter of minutes to hours (6). For is one of the most cold-hardy temperate species known and has example, in the flesh fly, Sarcophaga crassipalpis, the first species long been a model for studying freeze tolerance (39). Larvae of in which RCH was described, exposure to 0 °C for as little as E. solidaginis are capable of RCH (40) although the capacity for 30 min significantly enhances cold tolerance at −10 °C (6). RCH RCH in isolated tissues has not been addressed in this species. has since been described in dozens of insect species (7), including When midgut and salivary gland tissue from winter-acclimatized both freeze-intolerant (insects in which internal ice formation is third instar larvae were removed and directly frozen at −20 °C, lethal) and freeze-tolerant species (insects that tolerate internal high mortality resulted. Cell survival of midgut tissue dropped to ice formation) (8, 9). Naturally occurring thermoperiods can elicit RCH (10), and RCH preserves essential functions such as courtship and mating (11, 12), supporting the relevance of this 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., and process to natural populations. D.L.D. wrote the paper. Although the ecological relevance of RCH has been estab- The authors declare no conflict of interest. lished, the physiological mechanisms are poorly understood. Data deposition: The sequences reported in this paper have been deposited in the Gen- RCH results in a slight increase in the cryoprotectant glycerol (6, – fl – Bank database (accession nos. KC810053 KC810056). 13), changes in cell membrane composition and uidity (14 16), 1 To whom correspondence may be addressed. E-mail: [email protected] or denlinger.1@osu. and up-regulation of a single heat shock protein in the brain (17). edu. However, gene expression does not appear to be a major driver This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of RCH; in the flesh fly, Sarcophaga bullata, no transcripts (out 1073/pnas.1306705110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306705110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 ∼50% (Fig. 1A) whereas that of the salivary gland was ∼60% (Fig. 1B), in contrast to untreated controls held at 4 °C, which had >90% survival (Fig. 1 A and B). However, when tissues were ﬁrst given a 1-h RCH period before freezing, in which temperature was gradually lowered from 4 °C to −20 °C, survival increased by 25–30% in each tissue (Fig. 1 A and B). This cold- hardening response occurred ex vivo in the absence of stimulation from nerves or hormones, indicating that RCH in tissues of E. 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 in intracellular calcium in tracheal cells of E. solidaginis when temperature was lowered from 25 °C to 0 °C at 1 °C/min (Fig. 1 C and D). Lowering temperature from 25 °C to 20 °C raised intracellular calcium by 7%, and calcium rose continuously as temperature decreased. Maintaining temperature at 25 °C for the duration of the 40-min experiment failed to elicit a signiﬁcant increase in cytosolic calcium (Fig. 1E). Cold-induced increases in cytosolic calcium have also been demonstrated in plants (35) although cold exposure in plants causes a single, sharp spike in intracellular calcium, suggesting that a different mechanism is at play. Modest elevation of intracellular calcium at low temperature is also known in ﬁsh (41) and mammals (42, 43) although not in the context of cold hardening. In insects, disruption of ion gradients during chilling is responsible for the loss of neuromuscular function at low temperature (26–33), but whether these ion movements are used to trigger cold hardening has not been addressed.

RCH Activates Calcium-Dependent Signaling Pathways. Downstream of calcium entry into the cell, chilling activated calcium-dependent signaling pathways in E. solidaginis. Specifically, we monitored the activity and phosphorylation state of the calcium-dependent signaling enzyme calcium/calmodulin dependent protein kinase II (CaMKII) in response to low temperature. CaMKII is a multi- functional signaling enzyme that regulates numerous metabolic and gene expression processes (44), and whereas this enzyme has been linked to cellular stress in mammalian systems (e.g., ref. 45), it has not been associated with environmental stress. In larvae of E. solidaginis, RCH conditions induced a significant increase (from − − 0.65 to 0.88 pmol·min 1·μg protein 1) in the activity of CaMKII (Fig. 1F). Although this protein was activated during RCH, after prolonged freezing at −15 °C for 24 h, with and without 2 h recovery at 18 °C, CaMKII activity decreased to levels that were indistinguishable from both control and RCH-treated larvae (Fig. 1F). Thus, activation of this protein occurred specifically during the RCH period. Additionally, RCH decreased phosphorylation at the inhibitory Thr306 residue by 60% (Fig. 1 G and H), which is consistent with enzyme activation (44). In contrast, chilling at 4 °C for 24 h and 2 h recovery from freezing significantly increased the phosphorylation ratio (Fig. 1 G and H), suggesting deactivation of Fig. 1. Rapid cold hardening reduces freezing injury in tissues of the this enzyme in response to prolonged chilling and recovery from fl goldenrod gall y, E. solidaginis and activates calcium-signaling pathways. freezing. We cloned and sequenced full-length calmodulin (Gen- (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 Bank accession no. KC810053) and CaMKII (GenBank accession −20 °C. (C–E) Intracellular calcium as a function of temperature in tracheal no. KC810054) transcripts from E. solidaginis and measured their cells of E. solidaginis.(F) Activity of calcium/calmodulin-dependent protein tissue-specific expression. Predicted amino acid sequences for kinase II (CaMKII) following RCH, freezing for 24 h, and freezing with 2 h both proteins indicated that this signaling axis is highly conserved recovery. (G) Levels of both total CaMKII and phospho-Thr306 CaMKII in across insects and mammals (Figs. S1A and S2A), and transcripts response to low temperature. (H) The ratio of phospho-Thr306 CaMKII to were expressed in all six larval tissues tested (Figs. S1B and S2B), total CaMKII in response to low temperature. In A and B, “Control” tissues were held at 4 °C for 3 h, “Directly Frozen” tissues were directly transferred suggesting that these genes could mediate cold sensing throughout from 18 °C to −20 °C for 2 h whereas “RCH” tissues were gradually chilled the body. from4°Cto−20 °C over 1 h, then held at −20 °C for 2 h. In C, relative calcium concentration of a representative tracheal end cell is indicated by the provided color scale. In D and E, an asterisk indicates a significant difference activity and phosphorylation were measured in whole-body protein extracts. between a particular time point and the initial calcium concentration (re- All values are mean ± SEM, n ≥ 4, with different letters indicating significant peated measures ANOVA, post hoc Bonferroni, P < 0.05). In F–H, CaMKII differences (ANOVA, Tukey, P < 0.05).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306705110 Teets et al. Downloaded by guest on September 30, 2021 Functional Significance of Calcium Signaling During RCH. To de- termine the functional significance of calcium signaling during cold sensing, we used pharmacological inhibitors to block various com- ponents of calcium-signaling pathways during RCH. In midgut and salivary gland tissues of larval E. solidaginis, removing calcium from the medium, blocking calcium channels with LaCl3, and chelating intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid, acetoxymethyl ester (BAPTA-AM) all inhibited ex vivo RCH, reducing survival 17–47% relative to tissues incubated with calcium (Fig. 2). In addition, blocking calmodulin with the drug N-(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 following RCH (Fig. 2). However, inhibition of inositol triphosphate (IP3)- mediated calcium signaling had no effect on survival, suggesting that IP3-mediated calcium release is not involved in cold sensing. These drugs were not toxic to tissues held at 4 °C and had little effect on the survival of tissues directly frozen at −20 °C (Fig. S3 A and B). Furthermore, at milder temperatures, inhibition of calcium signaling reduced baseline freezing tolerance. After direct freezing at −17.5 °C, cell survival was significantly higher than at −20 °C (71.1% vs. 51.1% for midgut, 85.9% vs. 60.5% for salivary gland; Fig. S3 C and D), but inhibition of calcium signaling reduced survival at −17.5 °C (Fig. S3 E and F). These pharmacological data demonstrate that calcium entry is not simply a symptom of low temperature but is an essential cold-sensing mechanism mediating rapid responses to cold. PHYSIOLOGY

Calcium Signaling also Mediates RCH in a Freeze-Intolerant Insect. The above results clearly demonstrate a role for calcium signaling during RCH in larvae of E. solidaginis, a freeze-tolerant or- ganism. Subsequent experiments indicated that calcium signaling also governs cold sensing and RCH in a freeze-intolerant species, adults of the flesh fly S. bullata. Salivary gland tissues from this species were more amenable to calcium imaging, allowing a more in-depth analysis of the dynamics of cold-induced calcium movements. Chilling to 0 °C elicited a 70% increase in intracellular calcium in adult salivary gland tissue (Fig. 3 A and B) whereas maintaining tissues at 25 °C for the duration of the experiment failed to evoke a calcium response (Fig. 3C). When the cooling rate was increased to 2 °C/min, calcium concentrations showed a similar increase although the concentration at a given temperature 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- Fig. 2. Pharmacological inhibition of calcium signaling blocks rapid cold plain the rapid attenuation of RCH upon warming (46). hardening in tissues of E. solidaginis.(A) Fluorescent cell viability images of Like E. solidaginis, RCH in isolated tissues of S. bullata midgut tissue incubated with the indicated drug at the following temperature conditions: Control, 4 °C/3 h; Directly Frozen, direct transfer to −20 °C was dependent on calcium-signaling pathways. For these experi- − − ments, RCH was generated by directly transferring tissues to 0 °C for 2 h; RCH, slow ramp from 4 °C to 20 °C over 1 h, then held at 20 °C for 2 h. Green nuclei represent live cells, and red nuclei represent dead cells. for 2 h, before transferring tissues to the discriminating temper- fi − (B and C) Quanti ed viability results for (B) midgut and (C) salivary gland ature ( 14 °C) for 2 h. This stepwise protocol is the typical ap- tissue. All values are mean ± SEM, n = 4, and different letters indicate sig- proach for demonstrating RCH in the laboratory (7). In both nificant differences among the three temperature treatments where an midgut and fat body tissue from adults of S. bullata, blocking asterisk indicates a significant reduction in survival in the RCH group relative calcium entry and calmodulin activation prevented RCH (Fig. 3 to tissues incubated with calcium present (ANOVA, Tukey, P < 0.05). NCF, F–H) whereas these drugs had no effect on control tissues and nominally calcium free medium. LaCl3 is a general calcium channel blocker, tissues directly cold shocked at −14 °C (Fig. S3 G and H). In this BAPTA is an intracellular calcium chelator, W-7 is an antagonist of the cal- species, calmodulin (GenBank accession no. KC810055) and cium binding protein calmodulin, and KN-93 inhibits activation of CaMKII whereas 2-APB is an inhibitor of IP -mediated calcium signaling. CaMKII (GenBank accession no. KC810056) were expressed in 3 all postembryonic developmental stages and in nearly every tissue (Figs. S4 and S5), suggesting that this signaling axis could mediate of the cell physiology of insect low temperature response. A hy- cold sensing throughout the body in every developmental stage. pothetical model for the mode of calcium entry and the down- Thus, although the capacity for freeze tolerance has evolved stream targets of calcium signaling in response to low temperature numerous times in the insect lineage (47), cold-induced calcium is presented in Fig. 4. Low temperature directly inhibits ATP-de- signaling appears to be a general response to chilling in insects. + + pendent calcium-exporting functions, such as the Na /Ca2 - + + Conceptual Model for Calcium-Mediated Cold Sensing. The results exchanger coupled to Na /K -ATPase, causing a gradual leak of presented here represent a significant advance in our understanding calcium ions into the cell (33). This mechanism of cold-induced

Teets et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 3. Calcium signaling also mediates cold sensing and rapid cold hardening in a freeze-intolerant fly, S. bullata. Intracellular calcium concentration in salivary 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 by warming 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 viability results for (G) midgut and (H) fat body tissue. Values are mean ± SEM, n = 3for(B–D), n = 2for(E), n = 4for(G and H). In A, relative calcium concentration of a representative segment of salivary gland tissue is indicated by the provided color scale. In B–E, an asterisk indicates a significant difference between a particular time point and the initial calcium concentration (repeated measures ANOVA, post hoc Bonferroni, P < 0.05). In G and H, an asterisk indicates a significant reduction in survival in the RCH group relative to 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 calcium binding protein calmodulin, and KN-93 inhibits activation of CaMKII whereas 2-APB is an inhibitor of IP3-mediated calcium signaling.

calcium influx is different from cold sensing in the nervous system, calcium-dependent enzyme apoptosis signal-regulating kinase I which involves direct activation of excitable, cold-sensitive transient regulates p38 MAP kinase signaling (52), thus providing a poten- receptor potential channels in sensory neurons (48) that carry the tial link between calcium and p38 signaling (20) during RCH. signal to specific regions of the brain (49). Although we cannot rule Calcium signaling provides a direct, intuitive means by which out that RCH is simply responding to cold injury, with calcium isolated insect tissues detect and respond to low temperature. influx as the token symptom, the observation that calcium levels Although the sensing mechanisms governing behavioral responses closely track environmental temperature (Fig. 1 C and D and 3 B– to low temperature in the nervous system are well-established E) suggests a general cold-sensing role for calcium. Although the (48), cold-sensing mechanisms in nonnervous tissues have been downstream targets of calcium signaling during RCH have not hitherto unexplored. Understanding the mechanisms by which been established, calcium signaling interacts with a number of insects tolerate low temperature is essential for predicting their processes known to be important for cold hardening, including response to a changing climate (53). Furthermore, mechanisms apoptosis signaling (22, 23) and carbohydrate mobilization (50). In of cold hardening can be exploited to manipulate populations of the cold hardy atsugari mutant of D. melanogaster, increased in- insect pests (54). Our group has recently explored the possibility tracellular calcium improves cold tolerance by boosting mito- of disrupting overwintering diapause to control pest populations chondrial metabolism at low temperature (38), and subsequent (55), and we envision disruption of acute cold hardening to be an experiments will test whether metabolic changes associated with equally effective strategy. RCH (e.g., 13, 18, 51) are indeed calcium-regulated. Also, although the present study addressed only the calcium/calmodulin/ Methods CaMKII signaling axis, several other calcium-dependent signaling Animals. Galls containing third instar larvae of E. solidaginis, located on the pathways are known to mediate stress responses. For example, the stems of senesced Solidago canidensis plants, were collected from various

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306705110 Teets et al. Downloaded by guest on September 30, 2021 Fluorescence was recorded in a region of interest that consisted of a single tracheal end cell in E. solidaginis and four to five salivary gland epithelial cells in S. bullata. Intracellular calcium concentration was calculated according 2+ 2+ the following equation: [Ca ]i = Kd(F/Fo)/(Kd/[Ca ]i-rest + 1 − F/Fo), where F is 2+ the fluorescence, Fo is the initial fluorescence, and [Ca ]i-rest is the resting calcium 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 according to Woodruff et al. (59).

CaMKII Assays. CaMKII activity was measured with the Signatect CaMKII Assay Kit (Promega). In this assay, CaMKII transfers phosphate from [γ-32P]ATP to a synthetic substrate for CaMKII, which is immobilized on a biotinylated membrane and measured with a scintillation counter. Protein was extracted from whole larvae of E. solidaginis with radioimmunoprecipitation assay buffer 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 same manner, and 35 μg of protein from each sample was loaded into a 4–15% gradient SDS/PAGE gel. Western blotting was conducted according to Yi et al. (23). Rabbit anti-CaMKII (total) was obtained from Santa Cruz Bio- technology and rabbit anti-CAMKII (pThr305) was obtained from Millipore whereas mouse anti-β-tubulin was obtained from the Developmental Stud- ies Hybridoma Bank. Densitometry was conducted with AlphaView SA software (ProteinSimple), with each sample normalized to β-tubulin. Each Western blot was repeated with six independent biological replicates. To verify whether mammalian-based assays and antibodies would work, we cloned and characterized full-length calmodulin and CaMKII cDNA sequences using the Clontech SMARTer RACE kit (Clontech Laboratories). ﬁ

Additionally, in E. solidaginis, we measured tissue speci c expression in PHYSIOLOGY larval brains, midguts, fat body, salivary glands, Malpighian tubules, and Fig. 4. Working model for the role of calcium signaling during cold sensing epidermis using RT-PCR (Figs. S1 and S2). In S. bullata, we measured both fi fi and rapid cold hardening. Low temperature causes an increase in intracellular developmental and tissue-speci c expression pro les (Figs. S4 and S5). calcium concentration, and we hypothesize that this calcium influx occurs by Primer sequences for RACE and PCR detection of transcripts are provided low temperature inhibition of ATP-dependent calcium export mechanisms in Table S1. coupled with calcium entry through calcium leak channels (CLC). In this model, low temperature inhibits the activity of both sarcoplasmic endo- Cell Viability Assays. Tissues were dissected in Coast’s solution at room plasmic reticulum calcium ATPase (SERCA) and sodium/potassium ATPase temperature. Tissues from E. solidaginis were exposed to the following (Na/K ATPase) coupled to the sodium calcium exchanger (NCX). Inside the cell, temperature conditions: control (4 °C, 3 h), directly frozen (directly trans- calcium, via calcium/calmodulin-dependent protein kinase II (CaMKII) and ferred from room temperature to −17.5 or −20 °C, and held there for 2 h), other unknown mechanisms, triggers pathways involved in rapid cold hard- and RCH (slowly ramped from 4 °C to test temperature over 1 h, then held ening, thereby enhancing the cell’s cold tolerance. Dashed lines and arrows at test temperature for 2 h). For S. bullata, tissues were exposed to control indicate speculative relationships that were not experimentally determined (25 °C, 4 h), cold shock (−14 °C, 2 h), and RCH (0 °C 2h, −14 °C 2h) conditions; in the present study. all tissues remained supercooled at −14 °C. Cell viability was assessed with the LIVE/DEAD sperm viability assay (Life Technologies) (24), which is a membrane integrity assay consisting of SYBR green and propidium iodide. In this goldenrod fields in central and southwest Ohio from September to January assay, living cells with intact membranes fluoresce green whereas dead cells 2007–2012. Galls were stored at 18 °C, and larvae were removed just before with damaged membranes fluoresce red. Viability is expressed as the per- experimentation. Flesh flies, S. bullata, were laboratory-reared according to centage survival based on the counts of 300 cells per sample. To test whether Denlinger et al. (56) under nondiapausing conditions (25 °C, 16:8 light:dark). calcium signaling is essential for RCH, tissues were exposed to the following Adults were fed sugar and liver ad libitum, and males were used for ex- solutions and drugs to manipulate calcium signaling: nominally calcium free periments 4–8 d after eclosion. (NCF) medium, Coast’s solution prepared without calcium; 250 μM LaCl3, a general calcium channel blocker; 100 μM BAPTA-AM, an intracellular cal- Calcium Imaging. Tissues from E. solidaginis and S. bullata were dissected in cium chelator; 50 μM W-7, an inhibitor of calmodulin; 100 μM KN-93, an μ Coast’s solution (57) containing (in mM) 100 NaCl, 8.6 KCl, 4.0 NaHCO3,4.0 inhibitor of CaMKII, and 10 M 2-aminoethoxydiphenyl borate (2-APB), an NaH2PO4-H2O, 1.5 CaCl2-2H2O, 8.5 MgCl2-6H2O, 24 glucose, 25 Hepes, and inhibitor of IP3-mediated calcium signaling. Tissues were loaded with drugs 56 sucrose. For experiments with E. solidaginis, the Coast’s solution was for 30–60 min before conducting the same temperature experiments supplemented with 250 mM glycerol. After dissection, tissues were loaded described above. with 10 μM fluo-3 AM (Life Technologies) for 1 h at room temperature. The loading solution contained 0.2% pluronic F-127 (Life Technologies) to dis- Statistics. All data are expressed as mean ± SE. Data from calcium-imaging perse the dye. After loading, cells were rinsed twice in Coast’s solution, kept experiments were analyzed with repeated measures ANOVA and a post hoc in the dark at room temperature, and imaged within the next 3 h. Pre- Bonferroni test. All other means were compared with ANOVA and Tukey’s liminary experiments established that tracheal cells from E. solidaginis and post hoc multiple comparisons procedure in JMP9 (SAS Institute). For calcium- salivary gland cells from S. bullata were most amenable to calcium imaging. imaging experiments, n = 2–5 for each experiment whereas, for CaMKII assays, = – Other tissues were difficult to maintain in the proper focal plane or failed n 5 6 for each treatment. Cell viability assays were conducted with four to take up the calcium imaging dye. Tissues were imaged with a Visitech biological replicates per treatment, and data were arcsin square-root trans- Infinity3 Hawk 2D Array live-cell confocal microscope at the Ohio State formed before analysis. Campus Microscopy and Imaging Facility. Cells were excited at 488 nm, and fluorescence at 520 nM was measured every 10 s for the duration of the ACKNOWLEDGMENTS. We thank Sarah Cole, Brian Kemmenoe, Richard experiment. Temperature was controlled with an Instec TSA02i inverted Montione, and other members of the Ohio State Campus Microscopy and Imaging Facility for assistance with these experiments. We also thank microscope thermal stage (Instec). Although images were acquired every Dr. Michael Ibba for providing laboratory space for experiments requiring 10 s, low temperature caused a slight drift in the focal plane in most sam- radiolabeled substrates, Josh Stapleton for help with densitometry, and ples, necessitating periodic refocusing. As a result, samples were refocused members of the Laboratory for Ecophysiological Cryobiology at Miami every 2.5–5 min, so results are presented only in 2.5- to 5-min increments. University for assistance with gall collecting. Peter Piermarini and Larry

Teets et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Phelan, Ohio State University, kindly provided comments on a draft of this and John Duman, University of Notre Dame, for critically reviewing the paper. manuscript. We acknowledge Brent Sinclair, University of Western Ontario, This work was supported by National Science Foundation Grant IOS-0840772.

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