Chaperone proteins and winter survival by a freeze tolerant insect

Journal of Insect Physiology 57 (2011) 1115–1122

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Chaperone proteins and winter survival by a freeze tolerant insect§ Guijun Zhang, Janet M. Storey, Kenneth B. Storey * Institute of Biochemistry and Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 November 2010 Received in revised form 24 February 2011 Accepted 25 February 2011

The role of chaperone proteins in the winter survival of insects was evaluated in freeze tolerant gall fly larvae, Eurosta solidaginis. Levels of four heat shock proteins (Hsp110, Hsp70, Hsp60, Hsp40), two glucose-regulated proteins (Grp75, Grp78) and three others (tailless complex polypeptide 1 [TCP-1], aAcrystallin, aB-crystallin) were tracked in outdoor larvae from September to April and, in addition, laboratory experiments assessed chilling, freezing, and anoxia effects on these proteins. Gall fly larvae showed consistent elevation of Hsp110, Hsp70, Hsp40, Grp78 and aB-crystallin over the late autumn and winter months, generally 1.5–2.0-fold higher than September values. This suggests that these proteins contribute to cell preservation over the winter months via protection and stabilization of macromolecules. By contrast, levels of the mitochondrial Hsp60 fell to just 40% of September values by midwinter, paralleling the responses by numerous mitochondrial enzymes and consistent with a reduction in total mitochondria numbers over the winter. None of the proteins were altered when 15 8C acclimated larvae were chilled to 3 8C for 24 h but Hsp70, Hsp40 and Grp75 increased during freezing at 16 8C for 24 h whereas others (Hsp110, TCP-1 and both crystallins) increased significantly after larvae thawed at 3 8C. Anoxia exposure (24 h under N2 gas at 15 8C) elevated levels of Hsp70, Grp78 and the two crystallins. Levels of active hyperphosphorylated heat shock transcription factor (HSF1) were also analyzed, giving an indication of the state of hsp gene transcription in the larvae. HSF1 was high in September and October but fell to less than 40% of September values in midwinter consistent with suppression of gene transcription in diapause larvae. HSF1 levels responded positively to freezing and increased robustly by 4.9-fold under anoxia. Overall, the data provide strong evidence for the importance of protein chaperones as a mechanism of cell preservation in freeze tolerant insects. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Eurosta solidaginis Heat shock proteins Glucose-regulated proteins Cytoprotection Cold hardiness Anoxia

1. Introduction The field of insect cold hardiness, like many subdisciplines of comparative biochemistry and physiology, is largely driven by the interests and inventiveness of individual scientists, each following a different trail to drive the field forward. Karl Erik Zachariassen spearheaded one of these trails with a focus on how insects managed ice, water and ions at subzero temperatures, making major discoveries about the nature of ice-nucleating versus antifreeze proteins and about the mechanisms used to sustain and/or re-establish ionic gradients during subzero or freeze–thaw exposures. These he published in many insightful articles (recent reviews include Zachariassen and Kristiansen, 2000; Zachariassen et al., 2004). Like the other prominent Viking physiologists of the

§ Note: this is a contribution to the memorial special issue for KE Zachariassen. Abbreviations: HSP, heat shock protein; HSF, heat shock factor; GRP, glucoseregulated protein; TCP, tailless complex polypeptide; CCT, chaperonin containing t-complex polypeptide-1; UPR, unfolded protein response. * Corresponding author. Tel.: +1 613 520 3678; fax: +1 613 520 3749. E-mail address: [email protected] (K.B. Storey).

0022-1910/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.02.016

twentieth century (including August Krogh, Per Scholander, Torkel Weis-Fogh, Knut Schmidt-Neilsen and Kjell Johansen), Zachariassen sought to define principles and embraced comparative models (with requisite trips to exotic locales) to elucidate Nature’s design principles. This led him to broader studies of water balance in insects with both cold-hardy and desert model insects and also into ecotoxicology and the methods of dealing with toxic ions such as cadmium. One of us (KBS) had the great good fortune to share cigars with Karl Erik on three different continents, meeting him in both his northern plumage (amongst ice flows) and in his more southerly persona in Africa and sharing research adventures and tall tales. Research in the Storey lab has followed a different trail to seek out principles of insect cold hardiness by focusing mainly on cryoprotectant metabolism (low molecular weight sugars and polyols), cellular energetics, enzymology and signal transduction (for review Storey, 1990, 1997; Storey and Storey, 1991, 1992, 2010). Both the Zachariassen and Storey labs actually shared an ultimate goal of identifying the principles and mechanisms of cell preservation and viability extension that allow insects to survive for many months at subzero temperatures, even though our focus was on different adaptive strategies. The present article focuses on

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a mechanism of cell preservation that would have intrigued Karl Erik because of his interest in proteins that protect organisms from damage at subzero temperatures. A ubiquitous and marked cell preservation response to a wide range of environmental stresses is the up-regulation of a group of highly conserved proteins, the so-called heat shock proteins (HSPs) due to their initial discovery as responses to acute high temperature exposure. The major families of these proteins are defined by their molecular masses: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and the small Hsps (sHsps) (sizes > 30 kDa) (Gething and Sambrook, 1992; Fink, 1999; Feder and Hofmann, 1999). Note, however, that revised nomenclature for the mammalian proteins is now beginning to take hold and calls these families HSPH, HSPC, HSPA, HSPD, DNAJ, and HSPB, respectively, without a molecular mass designation (Kampinga et al., 2009). HSP families have both constitutive and stress-inducible members and most act as chaperones, either alone or in cooperation with partners, to prevent the aggregation of unfolded proteins (either naı¨ve or denatured), facilitate folding of naive proteins or re-folding of malfolded proteins, and aid in intracellular protein trafficking and assembly (Gething and Sambrook, 1992; Feder and Hofmann, 1999). Interestingly, despite the thousands of studies in the literature that have evaluated HSP responses to heat and many other stresses, there has been relatively little investigation of HSP involvement in organismal response to cold. Furthermore, most studies that have analyzed cold stress have focused on species that are not cold hardy so that very little is known to date about the role of chaperones in natural cold tolerance. For example, among insects, both the first analysis of cold exposure on Hsp70 expression (Burton et al., 1988) and the most recent study that analyzed the expression of 11 hsp genes (8 were up-regulated during recovery after cold exposure) (Colinet et al., 2010) focused on cold stress in Drosophila melanogaster. Only a few studies have examined cold hardy species (summarized by Clark and Worland, 2008). For example, Sonoda et al. (2006) reported up-regulation of Hsp90 during cold acclimation in nondiapausing larvae of the rice stem borer, Chilo suppressalis, but not in diapausing larvae that already had elevated levels of the protein. A series of studies has also documented up-regulation of hsp70, hsp60 (chaperonin) and tcp1 (t complex polypeptide-1) genes in cold hardy pupae of the onion maggot, Delia antiqua (Kayukawa et al., 2005; Chen et al., 2006; Kayukawa and Ishikawa, 2009). Studies of the Antarctic flightless midge, Belgica antarctica, showed continuous upregulation of hsp genes (hsp70, hsp90 and small hsps) in the cold hardy larval stage, but not in the short-lived summer adult stage (Rinehart et al., 2006). Up-regulation of several hsp genes is now known to be component of diapause in multiple orders of insects (Rinehart et al., 2007) and many insects are in diapause over the winter months so this implies that HSP chaperones could be important contributors to cold-hardiness and life extension over the winter months of cold dormancy. Indeed, pupae of the flesh fly, Sarcophaga crassipalpis, up-regulate Hsp70 and Hsp23 during diapause and these are both responsive to cold and heat stress in non-diapausing flesh flies (Yocum et al., 1998; Rinehart et al., 2000). RNAi knockdown of these proteins reduced the ability of diapausing pupae to survive cold, but did not alter their capacity for diapause so this suggested that these two Hsps may have a primary function in cold tolerance (Rinehart et al., 2007). In the present study, we evaluate the responses of multiple types of chaperone proteins in the seasonal cold tolerance of the goldenrod gall fly larvae, Eurosta solidaginis Fitch (Diptera, Tephritidae), a well-studied model of insect freeze tolerance (for review Storey, 1990, 1997; Storey and Storey, 1991, 1992, 2010). Responses were tracked over the winter months and, in addition, laboratory experiments assessed chilling, freezing, and anoxia

effects on protein levels. The proteins assessed were Hsp110, Hsp60, Hsp40, three members of the HSP70 family (inducible Hsp70, Grp75, Grp78), tailless complex polypeptide 1 (TCP-1) that is involved in the folding of action and tubulin in the cytoplasm (Kayukawa et al., 2005), and two crystallins (aA-crystallin, aBcrystallin). Expression patterns of the heat shock transcription factor (HSF1) that regulates hsp gene expression were also analyzed. 2. Materials and methods 2.1. Animals Galls containing last instar larvae of E. solidaginis were collected during mid to late September 1999 from goldenrod plants in fields around the Ottawa area. Some galls were placed in cloth bags, hung on a fence (always above the snowline), and kept outdoors over the winter months. Galls were sampled in the second week of each month from September to April. At each sampling time, a group of galls was brought into the laboratory at 9:00 a.m. and briefly stored in an incubator set to the current outdoor temperature. Galls were opened as soon as possible and larvae were flash-frozen in liquid nitrogen and then stored at 80 8C. Other September-collected galls were brought into the laboratory and acclimated to 15 8C in incubators for two weeks. After this time, some galls were opened and larvae were sampled as above. The remaining galls were divided into two experimental groups and received either cold or anoxia exposures. For cold exposure, larvae in their galls were treated as follows: (a) acutely lowered from 15 8C to 3 8C for 24 h, (b) then acutely switched down to 16 8C for another 24 h (a freezing stress), and (c) then acutely switched back to 3 8C for 24 h of thawed recovery. For anoxia exposure, 15 8C-acclimated larvae were first removed from their galls and placed in open petri dishes with a piece of moist filterpaper to prevent desiccation. The dishes were then placed in plastic containers with two valves on the top, one to introduce nitrogen gas and one to vent the gas. Containers were flushed with 100% nitrogen gas for 20 min and then both valves were sealed and containers were replaced at 15 8C. Animals were sampled after 4 or 24 h of anoxia exposure by rapidly opening a container, removing the petri plate and quickly freezing the larvae. 2.2. Sample preparation and protein content Whole frozen larvae (6 per sample) were weighed and quickly homogenized 1:2 (w/v) in ice-cold buffer that inhibited the activities of endogenous protein phosphatases and kinases: 20 mM HEPES, 400 mM NaCl, 20% (v/v) glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM NaF, 10 mM b-glycerophosphate, and 1 mM Na3VO4. Protease inhibitors were also added: 1 mM each of phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and benzamidine. Samples were centrifuged at 10,000  g at 4 8C for 10 min and supernatants were transferred to a fresh tube. Soluble protein concentration was determined by the Coomassie blue dye-binding method using the Bio-Rad prepared reagent with bovine serum albumin as the standard. Aliquots of supernatant were then mixed 1:1 (v/v) with 2 sample buffer containing 100 mM Tris–HCl pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 5% (v/v) b-mercaptoethanol and 0.2% (w/v) bromophenol blue. Samples were boiled for 5 min, chilled and then frozen at 80 8C for long term storage. 2.3. SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting SDS–PAGE gels containing 8, 10 or 12% acrylamide (depending on the molecular mass of the target protein) were prepared with 5%

G. Zhang et al. / Journal of Insect Physiology 57 (2011) 1115–1122

3. Results 3.1. Winter temperature profile Fig. 1 shows the daily maximum and minimum ambient temperatures for the Ottawa area (recorded at the Ottawa airport) from September 1, 1999 to April 30, 2000. The supercooling point of E. solidaginis larvae is about 8 8C and temperature profiles indicate that the larvae would experience daily minimum temperatures below 10 8C for long periods of December, January and February (as well as some periods in November and March). Maximum temperatures in many of these intervals were close to 0 8C (or even above) so that daytime thawing would occur. Therefore, the larvae would experience multiple freeze–thaw cycles over the winter months.

Temperature, ºC

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20 10 0 -10 -20 -30 Sep

Oct Nov 1999

Dec

Jan

Feb

Mar 2000

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Fig. 1. Temperature profiles for the 1999–2000 winter season in Ottawa, Canada. Data are from Environment Canada (http://climate.weatheroffice.gc.ca/ climateData) for the Macdonald-Cartier airport in Ottawa that is less than 10 km from the collection sites or the outdoor winter holding site of the larvae. Larvae were sampled during the second week of each month from September to April.

3.2. Winter profiles of HSPs Antibodies were initially tested to show their specificity and ability to detect insect proteins using 2D electrophoresis to show that the antibody crossreacted with a single spot at the expected molecular mass and isoelectric point of the target protein (data not shown). Subsequently, conventional one-dimensional SDS–PAGE and immunoblotting were used to analyze protein expression patterns under different treatment conditions. Hsp110, Hsp70 (the inducible form, also known as Hsp72), Hsp60 and Hsp40 as well as HSF-1 were all detected in E. solidaginis; in every case, the antibody used cross-reacted with a strong protein band at the expected molecular mass. Fig. 2 shows the relative levels of the four Hsps in the larvae from mid-September through to mid-April (larvae were sampled in the second week of each month). Levels of three proteins increased significantly over the midwinter months. Compared with amounts in September, the relative expression of Hsp110 increased in October by 1.60-fold and remained high in December, February and March (1.79, 1.69 and 1.4-fold higher than September values, respectively). Levels decreased again in April to 92% of the September value. Hsp70 showed an upward trend in October and levels were significantly higher than September values in December, February and March (1.25, 1.42, and 1.33-fold higher, respectively) but fell again in April. Hsp40 showed a comparable pattern with expression levels that were significantly

Eurosta solidaginis HSPs a

2.0

Relative protein levels

stacking gels. Equal amounts of protein were loaded into each well (10 or 20 mg depending on the target protein) and electrophoresis was carried out on a Bio-Rad mini-gel apparatus at 180 V for 40– 50 min with 1 running buffer (3.03 g Tris base, 14.4 g glycine, 1 g SDS per liter, pH 8.3). Subsequently, gels were immersed in transfer buffer (25 mM Tris pH 8.5, 192 mM glycine, 20% (v/v) methanol) for 10 min and then electroblotted onto polyvinylidene difluoride (PVDF) membrane (Millipore) by wet transfer with prechilled transfer buffer at 180 mA for 1.5 h at 4 8C. Membranes were then blocked with 2.5% non-fat milk in TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) at 21 8C for 20–45 min (for different target proteins). After rinsing with TBST, membranes were then incubated with primary antibody on a shaking platform for 2–3 h at 21 8C or overnight at 4 8C. Blots were then washed twice with TBST for 10 min, incubated with secondary antibody in TBST according to manufacturer’s instructions for 1–2 h at 21 8C, and then washed for 2  20 min with TBST. Most primary antibodies were purchased from Stressgen (catalogue numbers in brackets): Hsp110 (SPA-1103, rabbit polyclonal against a portion of the hamster protein), Hsp70 (SPA-812, rabbit polyclonal against recombinant human protein), Hsp60 (SPA-805, rabbit polyclonal raised against Hsp60 from an insect, Heliothis virescens), Hsp40 (SPA-400, rabbit polyclonal against recombinant human protein), HSF1 (SPA-901, rabbit polyclonal against recombinant human protein), Grp78 (SPA-826, rabbit polyclonal against recombinant human protein) and TCP-1 (CTA-191, rat monoclonal raised against recombinant mouse protein). Grp 75 (sc-1058), aAcrystallin (sc-22390) and aB-crystallin (sc-22391) were from Santa Cruz. Biotechnology, Inc. (all goat polyclonals raised against an epitope of the human protein). Blots were developed using the SuperSignal West Pico Chemiluminescent substrate (Pierce) according to the manufacturer’s protocol. Bands were visualized using a ChemiGenius (Syngene, MD, USA) and band densities were quantified using the associated GeneTools software. After immunoblotting was complete, PVDF membranes were stained for protein using Coomassie blue. On each blot, a strong Coomassie-stained band that did not change in intensity across control and experimental lanes was chosen and densities of these bands were also quantified. Immunoblot band intensities in each lane were then normalized against the corresponding intensity of the Coomassie blue stained band in the same lane to correct for any minor variations in sample loading. Data for n = 4 separately prepared samples (n = 6 larvae pooled per sample) were collected for each sampling point. Significance testing used analysis of variance with a post hoc Dunnett’s test to search for difference in the normalized band intensities between groups. For convenience in visually assessing the data in figures, data were plotted relative to the values for the September or control groups that were standardized to 1.0.

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a

a

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Hsp110 Hsp70 Hsp60 Hsp40

a a

b

b

a

a

a

1.0 a

0.5

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Oct

Dec

a a

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Fig. 2. Expression of Hsp110, inducible Hsp70, Hsp60 and Hsp40 proteins from September to April in freeze-tolerant E. solidaginis larvae. Data are means  S.E.M. (n = 4). a: Significantly different from the corresponding September value as determined by analysis of variance followed by a two-tailed Dunnett’s t-test, P < 0.01; b: P < 0.05.

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higher than September values in October, December, February and March (by 1.56, 1.62, 1.35 and 1.44-fold, respectively) and reduced again in April. However, the mitochondrial Hsp60 was unique in showing an opposite pattern of response over the winter months. Hsp60 levels declined during the winter to values that were 48%, 42% and 51% of September values in December, February and March but rose again in April.

the levels of any of the four proteins was seen after 4 h of anoxia exposure. However, after 24 h of anoxia, Hsp70 had responded robustly with levels rising to 1.5-fold over control values. Levels of the other HSPs were unchanged (Hsp40) or suppressed (to 58 and 62% of controls for Hsp110 and Hsp60, respectively) by 24 h anoxia exposure. 3.4. Winter profiles of other chaperones

3.3. HSP responses to laboratory cold and anoxia exposures Fig. 3A shows the responses of the Hsp110, Hsp70, Hsp60 and Hsp40 to cold and freeze/thaw of the larvae; the conditions are (a) controls acclimated at 15 8C, (b) larvae shifted acutely from 15 8C down to 3 8C and cold-exposed for 24 h, (c) larvae shifted acutely from 3 8C down to 16 8C and frozen for 24 h, and (d) larvae transferred back to 3 8C and sampled after 24 h thawed. None of the proteins were affected by the temperature drop from 15 to 3 8C and Hsp60 was not affected by any temperature change. Hsp110 expression levels were also unchanged by freezing exposure at 16 8C but levels increased significantly by 1.46-fold after the larvae were thawed at 3 8C for 24 h. By contrast, both Hsp70 and Hsp40 responded strongly to freezing at 16 8C with levels rising by 1.43 and 1.50-fold, respectively, after freezing at 16 8C for 24 h. When larvae were thawed at 3 8C for 24 h, Hsp70 levels were reduced again (to 80% of control values) whereas Hsp40 remained elevated at 1.61-fold higher than controls. Fig. 3B shows the effect of exposure to a nitrogen gas atmosphere (at 15 8C) on the larvae. No significant change in

Eurosta solidaginis HSPs

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3.5. Responses other chaperones to laboratory cold and anoxia exposures b

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An acute drop in temperature from 15 8C to 3 8C for 24 h did not affect the levels of any of the five proteins analyzed and four were also unaffected by freezing at 16 8C (Fig. 5A). However, levels of Grp75 increased by 1.78-fold in frozen larvae but the protein decreased again when animals thawed. By contrast, thawing triggered increases in TCP-1, aA-crystallin and aB-crystallin which increased by 1.54, 1.46, and 1.63-fold over control values whereas Grp78 decreased significantly. Short term anoxia exposure had no significant effect on the levels of any of the five proteins but 24 h anoxia exposure triggered increases in three proteins (Fig. 5B). Levels of Grp78 increased by 1.43-fold and levels of aA-crystallin and aB-crystallin were both 1.8-fold higher than controls.

b

Eurosta solidaginis GRPs, TCP-1 & crystallins 2.5

1.0 a

b

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Fig. 3. Expression of Hsp110, Hsp70, Hsp60 and Hsp40 in E. solidaginis larvae in response to (A) low temperature exposures, or (B) anoxia exposure. To analyze the effect of temperature, larvae were sampled from 4 conditions: controls acclimated at 15 8C for 2 weeks, followed by an acute shift to 3 8C for 24 h, followed by an acute shift to 16 8C for 24 h (larvae froze), and then thawing at 3 8C for 24 h. To analyze the effect of anoxia, larvae were sampled from three conditions: controls acclimated for 2 weeks at 15 8C, and larvae exposed to a nitrogen gas atmosphere for 4 or 24 h at 15 8C. Other information as in Fig. 1.

Relative protein levels

Relative protein levels

2.0

Fig. 4 shows the changes in the levels of two GRPs (Grp75, Grp78) as well as TCP-1, aA-crystallin and aB-crystallin over the winter season in E. solidaginis. GRPs are so-called due to their initial discovery as proteins that were strongly up-regulated in cells cultured in a glucose free medium (Shiu et al., 1977) and are now classed as members of the HSP70 (HSPA) family. As compared with September, the level of Grp75 showed a rising trend in October and was significantly higher by 1.79-fold in December before declining again in February and returning to September values in March and April. Grp78 showed a stronger response with a sustained significant elevation from October to March; levels were 1.82, 1.93, 1.81, and 1.67-fold higher than September values in October, December, February, and March, respectively. Grp78 content decreased again in April. TCP-1 protein levels were significantly increased in October and December (both 1.40-fold over September values), but levels declined later in the winter. aA-crystallin levels were elevated only in March (1.71-fold higher than September) but aB-crystallin was generally higher over the winter with levels that were 1.55-fold higher than September values in October, December and April and peaked at 1.84-fold higher in March.

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Fig. 4. Expression of Grp75 (mitochondrial Hsp70 family member), Grp78 (endoplasmic reticulum Hsp70 member), TCP-1, aA-crystallin and aB-crystallin in E. solidaginis larvae over the winter months. Other information as in Fig. 1.

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Eurosta solidaginis GRPs, TCP-1 & crystallins

Eurosta solidaginis HSF1 1.4

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3.6. HSF-1 Fig. 6A shows the relative expression levels of activated hyperphosphorylated HSF-1, detected at 95 kDa, in E. solidaginis larvae from September through to April. September and October values were equivalent but in later months HSF-1 levels declined. Relative levels in December and February were just 34–36% of the September value but HSF-1 began to rise again in March and April to levels that were 51% and 61% of September values, respectively. Fig. 6B shows the responses of activated HSF-1 to freeze/thaw. Cold exposure at 3 8C resulted in a significant increase (35%) in the amount of activated HSF-1 expressed in the larvae and this rose further (59%) during freezing at 16 8C. However, after thawing (3 8C for 24 h) the amount of activated HSF1 was significantly reduced to 76% of the corresponding 15 8C control value; this was also an 50% decrease as compared with the level in frozen larvae (P < 0.05). Fig. 6C shows HSF-1 expression in response to anoxia. Activated HSF-1 levels were increased by 55% after 4 h anoxia exposure and then rose strongly to 4.88-fold higher than control values after 24 h of anoxia. 4. Discussion All of the proteins analyzed in the present study are known to act as chaperones and are members of the heat shock family although some are better known by alternate names. Recently there have been concerted attempts to standardize the nomenclature of these proteins and to do so without including molecular masses in the name (Kampinga et al., 2009). Based on the nomenclature developed for the heat shock genes in the human genome, the proteins analyzed here could also be designated as follows. The three members of the HSP70 family, Hsp70 (inducible

Feb

Mar

Apr

a a

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b

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24 h Anoxic

Fig. 5. Expression of Grp75 (mitochondrial Hsp70 family member), Grp78 (endoplasmic reticulum Hsp70 member), TCP-1, aA-crystallin and aB-crystallin in E. solidaginis larvae in response to (A) low temperature or (B) anoxia exposures. Other information as in Fig. 2.

Dec

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Relative protein levels

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Fig. 6. Relative levels of activated heat shock transcription factor 1 (HSF1) in Eurosta solidaginis larvae. (A) Changes over the months from September to April, (B) effects of exposures to low temperatures, and (C) effects of anoxia exposure. Note that levels of the activated form of HSF1 are expressed relative to the value for inactive HSF1 in the corresponding September or control situation. Experimental treatments and other information are as in Figs. 1 and 2.

Hsp72), Grp75, Grp78 (also known as BiP), are respectively HSPA1, HSPA9 and HSPA5. Designations for Hsp110, Hsp60 (also known as GroEL) and Hsp 40 are HSPH2, HSPD1 and DNAJB1, respectively, whereas TCP1 is called CCT1, the alpha subunit of the cytosolic chaperonin (CCT) complex. The two crystallins belong to the small heat shock protein family with aA and aB crystallin designated as HSPB4 and HSPB5, respectively. Three Hsps (Hsp110, Hsp70, Hsp40) are known to work together and have prominent roles in protein folding/refolding in cytoplasmic and nuclear compartments. The inducible Hsp70, like its cognate counterpart Hsc70, conducts the actual ATPdependent folding of proteins, the binding of ATP triggering a critical conformational change that leads to the release of the bound substrate protein (Fink, 1999). Hsp40 is the well-known partner protein of Hsp70 and acts by stimulating ATPase activity and activating the substrate binding of Hsp70 (Suh et al., 1999). These two are sometimes termed the holdase (Hsp40) and the foldase (Hsp70) (Winter and Jakob, 2004). Although long known to

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be triggered in conjunction with Hsp70 and Hsp40 and to confer thermal tolerance (Oh et al., 1997), it was only recently determined that the role of Hsp110 is to catalyze nucleotide exchange on Hsp70 to increase the rate and yield of Hsp70-mediated refolding of proteins (Dragovic et al., 2006). With a function similar to Hsp70/Hsc70, Hsp60 is the prominent chaperone in the mitochondrial compartment, where it operates together with its partner Hsp10 in the folding and assembly of proteins that are transported into these organelles (Cheng et al., 1989). Grp75 (also known as HSP70-9B, mortalin-2 or HSPA9) also occurs predominantly in mitochondria (Ran et al., 2000) where it facilitates the folding and assembly of proteins as they enter the organelle in conjunction with Hsp60 and Hsp10. Its induction by heat stress or transgenic over-expression increased nematode lifespan by 40% (Yokoyama et al., 2002) so it is possible that Grp75 might have a role in viability extension in hypometabolic states. Grp78 (BiP, HSPA5) resides in the endoplasmic reticulum (ER) where it has a central role in the folding and assembly of proteins that are destined for export or insertion into membranes (Lee, 2001). It is also a central sensor in the unfolded protein response (UPR), a coordinated group of actions that deals with stress-induced increases in the number of unfolded proteins in the ER. The UPR consists of three main actions that aim to restore homeostasis: up-regulation of chaperone production (mainly Grp78 and Grp95) to enhance folding capacity, reduction of protein delivery to the ER by inhibiting ribosomal activity and, if needed, stimulation of unfolded protein degradation (Schroder and Kaufman, 2005). TCP-1 (CCT1) is one subunit of the hetero-oligomeric cytosolic chaperonin named CCT (chaperonin containing t-complex polypeptide-1) that is the cytosolic version of mitochondrial Hsp60 (Kayukawa et al., 2005). Early studies indicated that actin and tubulin were the only substrates of CCT but recent work has found numerous other protein substrates (Horwich et al., 2007). Finally, crystallins (originally found in vertebrate lens) are divided into a, b and g families, and the a-crystallin group consists of two gene products, aA and aB crystallin, that are members of the small HSPs (Klemenz et al., 1991). Both a- and b-crystallins have chaperone actions; in particular, both aA and aB crystallin are highly effective in preventing the depolymerization of actin (Wang and Spector, 1996). The present study demonstrates a clear involvement by a number of chaperone proteins in the winter survival of freeze tolerant gall fly larvae. Protein levels of Hsp110, Hsp70, Hsp40, Grp78 and aB-crystallin were consistently elevated over the late autumn and winter months in the larvae, typically in the range of 1.5–2.0-fold higher than September values (Figs. 2 and 4). TCP-1 was also elevated in October and December and Grp75 was high in December. In the Ottawa area, the period from October to March is the time during which E. solidaginis larvae exhibit well-developed cold hardiness with high levels of cryoprotectants (Storey, 1990). Diapause in this species typically extends from late October through to early to mid-February and is followed by a period of cold quiescence until warming temperatures in April trigger rapid pupation. In general, then, the seasonal pattern of high chaperone protein levels in E. solidaginis covers not just the diapause period but also the full season of cold hardiness. This suggests that these proteins are a beneficial component of cold hardiness and freeze tolerance in this species. This agrees with recent studies by Rinehart et al. (2007) who showed that although hsp70 and hsp23 genes were upregulated when S. crassipalpis entered pupal diapause, RNAi knockdown of the expression of these genes did not affect diapause capacity but reduced cold survival of diapausing pupae. Enhanced chaperone levels over the winter months could contribute to life extension and cytopreservation over the extended period of time when larvae are in a hypometabolic

state. Indeed, recent studies are increasingly recognizing cytoprotective mechanisms such as antioxidant defenses and elevated chaperones as general features of hypometabolism in its many forms including hibernation, estivation and anaerobiosis (Storey and Storey, 2007). For freeze tolerant gall fly larvae a number of benefits of high constitutive chaperone levels could be envisioned. Firstly, during extended periods of time spent frozen, larvae experience oxygen deprivation and declining energy levels (ATP, arginine phosphate) (Storey and Storey, 1985). This is a time when ATP-expensive activities such as protein turnover must be minimized and hence mechanisms that protect and/or rescue existing proteins from denaturation, such as by the action of chaperones, are of increased importance. Interestingly, array screening for genes that were up-regulated during anoxia-induced hypometabolism in turtle brain also included those discussed above (inducible hsp70, hsp40, aB-crystallin) (Storey, 2007) as well as grp75 (discussed in the next paragraph). Secondly, winter conditions of deep cold (temperatures in the Ottawa area are often 30 8C or lower in midwinter) have the potential to cause cold denaturation of macromolecules and this could be dealt with best by constitutive chaperone defenses. Indeed, actin disassembly due to cold shock has been described (Upadhya and Strasberg, 1999) and recent studies have provided strong evidence of a critical role of chaperonin (CCT) in stabilizing actin at low temperatures in an insect. Nonhardy pupae of D. antiqua showed cold-induced depolymerization of actin but coldhardy pupae did not; this was correlated with enhanced transcript levels not only of tcp1 but of all subunits of chaperonin (CCT) in the cold-hardy larvae (Kayukawa and Ishikawa, 2009). Furthermore, if actin depolymerization was inhibited chemically by Latrunculin B administration to larvae, then the subsequent cold-hardiness of pupae was compromised. Coupled with our findings of elevated amounts of TCP-1 protein in E. solidaginis in October to December and an additional report of high tcp1 gene expression in cold-hardy S. crassipalpis pupae (Rinehart et al., 2007), the importance of chaperonin stabilization of the actin cytoskeleton against cold denaturation is indicated. Given the known action of both aA and aB crystallin in actin stabilization (Wang and Spector, 1996), elevated levels of aB crystallin over the winter months may augment the action of TCP-1/CCT in stabilizing the cytoskeleton over the winter months. Thirdly, a potential for freeze denaturation of proteins also exists. Freezing converts 65% or more of total body water into extracellular ice and this means that concentrations of dissolved molecules (ions, metabolites, proteins) in remaining intracellular liquid would rise by about 3-fold which could promote denaturation or aggregation of proteins. Physical protection of intracellular macromolecules during freeze dehydration is one important function of the natural polyol cryoprotectants accumulated by freeze tolerant species (glycerol and sorbitol in the case of E. solidaginis) (Storey, 1990) but the present study suggests that enhanced levels of chaperones that can prevent and/or reverse the denaturation, depolymerization or aggregation of proteins could also be an additional important aspect of cryoprotection over cycles of freeze/thaw. The effects of laboratory cold or freezing experiments on the activation of the HSF-1 transcription factor also support these ideas. Transfer of larvae from 15 8C to 3 8C increased the amount of activated HSF-1 (although HSPs did not increase) and the decrease to 16 8C further elevated transcription factor levels with the appearance in increased Hsp70 and Hsp40. This indicates that activation of HSF-1 in E. solidaginis is cold sensitive, probably leading to an elevation of hsp mRNA transcripts, but that translation of the transcripts awaits another signal such as a rise in temperature that allows protein synthesis to go forward under favourable thermal conditions or desperate conditions such as

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unexpected freezing. The former situation may actually be what occurs during the autumn in nature over cycles of cold nights and warmer days and is actually consistent with the pattern of CCT upregulation as a cold shock protein in S. cerevisiae described earlier (Somer et al., 2002). Under anoxic conditions at 15 8C, levels of activated HSF-1 had begun to increase after 4 h (55% higher than controls) and soared to 4.9-fold over controls after 24 h showing that HSF-1 was also highly responsive to anoxia stress in E. solidaginis; this was matched with a strong increase in Hsp70 levels after 24 h of anoxia exposure. Gall fly larvae readily survived 18 days under a nitrogen gas atmosphere at 13 8C (Storey and Storey, 1990) and this excellent anoxia tolerance would certainly aid long term freezing survival during the winter. Notably, the key to survival in all anoxia tolerant species is entry into a hypometabolic state where energy expenditures on protein turnover are minimized and where preservation of existing proteins becomes key to life extension throughout the stress period (Storey and Storey, 2007). Upregulation of chaperone proteins is proving to be an integral part of anaerobiosis in multiple animal groups. In contrast with the other HSPs, Hsp60 showed a unique response over the winter in E. solidaginis, with protein levels of this mitochondrial chaperone strongly suppressed between December and March (minimum levels were just 40% of September values) (Fig. 4). Notably, this pattern parallels that of the activities of multiple mitochondrial enzymes (e.g. citrate synthase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, malic enzyme, cytochrome c oxidase and enzymes of fatty acid oxidation) that are reduced by 50–65% over the winter months in E. solidaginis (Joanisse and Storey, 1994, 1996; McMullen and Storey, 2008). Furthermore, winter larvae have only about half of the mitochondrial DNA content of summer larvae (Levin et al., 2003). All of these data are consistent with a reduction in the total number of mitochondria in E. solidaginis larvae by about half during the winter. This could be a useful strategy for a species that spends much of the winter in an oxygen-restricted frozen state (McMullen and Storey, 2008) where mitochondria might be vulnerable to either physical damage from freeze/thaw or to oxidative insult resulting from major changes in oxygen availability over freeze/ thaw cycles. Note, however, that the relative amount of Hsp60 in each mitochondrion is probably not affected so that, for the remaining organelles, the function of Hsp60 in the folding and assembly of proteins entering the mitochondria would be preserved. Viewed in this context, the results for the other mitochondrial chaperone, Grp75, are very interesting. Grp75 showed a rising trend in October, a peak content in December (1.79-fold higher than in September) and then a return to near September values in February and March. Compared with the behaviour of Hsp60 and the other mitochondrial constituents mentioned above, this indicates a substantial relative increase in Grp75 levels in the mitochondria that remain in the larvae over the winter months. This would provide a relative enhancement of mitochondrial chaperone activity comparable to that seen for the cytoplasmic and ER chaperones discussed above. The association of Grp75 (also known as mortalin-2) with lifespan extension in nematodes and immortalization of tumor cells, apparently related to inactivation of the p53 tumor suppressor (Wadhwa et al., 2002) suggests that it may have a key role to play in preserving viability of diapausing larvae over the winter months. Laboratory studies examined potential triggers of chaperone production in E. solidaginis larvae. Chilling alone (a decrease from 15 to 3 8C) was not sufficient to trigger enhanced production of any of the nine chaperone proteins (Figs. 3a and 5a). However, freezing at 16 8C triggered significant increases in Hsp70, Hsp40 and Grp75 suggesting a need for enhanced cytoprotection of proteins in both cytoplasmic/nuclear and mitochondrial compartments in the frozen state. Expression of HSPs is typically greatest after a stress is

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reversed (e.g. when returned to normal after a heat shock) and four proteins actually showed this pattern with levels of Hsp110, TCP-1 and both crystallins elevated (each by about 1.5-fold) after the larvae thawed. Freeze–thaw is an ischemia-reperfusion event and a study by Bluhm et al. (1998) showed that overexpression of aB-crystallin enhanced microtubule integrity under conditions of simulated ischemia in rat neonatal cardiac cells. Taken together, these data suggest that overnight freeze/thaw episodes in the autumn or early winter season might be the trigger that stimulates the accumulation of chaperones proteins and that their action may be particularly important in recovery/repair of freeze-induced damage to macromolecules. For example, enhanced levels of TCP-1, aA and aB crystallins after thawing could strongly indicate a need to correct freeze-induced damage to the actin cytoskeleton of cells and suggest that these three proteins may act cooperatively in protecting and stabilizing the cytoskeleton in the cold. The results for TCP-1 actually fit well with a study of CCT in yeast. Somer et al. (2002) found that CCT is a cold shock protein in Saccharomyces cerevisiae; northern blots showed that mRNA transcript levels rose when yeast were given cold exposure at 4 8C but western blots showed that levels of CCT subunits did not actually increase until yeast were returned to a higher temperature (10 8C). Since actin and tubulin are major substrates for TCP-1, and cold injuries can depolymerize these cytoskeletal proteins, Somer et al. (2002) hypothesized that upregulation of TCP-1 is related to the reorganization of actin and tubulin monomers during recovery from cold stress. One consequence of whole body freezing in E. solidaginis is an interruption of oxygen supply to cells probably because high tissue ice halts the muscle movements that are needed to open/shut spiracles and ventilate tracheoles. Indeed, larvae frozen at 16 8C showed multiple metabolic indicators of anaerobic metabolism; ATP and energy charge decreased over time and lactate and alanine accumulated (Storey and Storey, 1985; Churchill and Storey, 1989). HSPs are induced by anoxia exposure in other species; for example, anoxia rapidly induced hsp70 gene expression in D. melanogaster (Ma and Haddad, 1997) and severe hypoxia triggered up-regulation of multiple hsps in S. crassipalpis (Michaud et al., 2011). The present data show that this is also true for E. solidaginis. Short term exposure (4 h) to a nitrogen gas atmosphere at 15 8C did not significantly change levels of any of the proteins in the larvae but the amounts of Hsp70, Grp78, and the two crystallins increased significantly by 1.5– 2-fold after 24 h of anoxia (Figs. 3b and 5b). These data suggest that low oxygen triggers may be involved in the freeze/thaw and seasonal responses by chaperone proteins in E. solidaginis larvae. The heat shock transcription factor (HSF-1) mediates the heat shock response by activating the transcription of hsp genes (and others) (Morimoto, 1998). Activation of HSF1 is a multi-step process that includes trimerization of inactive monomers, relocalization to the nucleus, and phosphorylation to achieve transcriptional competence (Sorger, 1991). Seasonal changes in the levels of activated HSF-1 in E. solidaginis showed an interesting pattern (Fig. 6a). HSF-1 expression was high in September and October and low over the other months ranging from a low of 34% of the September value in February to 61% in April. These data indicate that HSF-1 action in stimulating hsp gene expression is high during the autumn months, leading to the rise in HSP protein levels that is first apparent in October. The subsequent suppression of HSF-1 over midwinter is consistent with a reduction in gene expression in the diapause state even though HSP proteins are sustained at high levels over this time. This disjoint between low levels of activated HSF-1 and high levels of the HSPs probably has one of two origins: (a) HSP expression levels in midwinter are sustained by hsp transcripts that are transcribed before the larvae enter cold diapause and these transcripts are probably stored in stress granules, or (b) perhaps more likely, once HSPs are synthesized they have very long half-lives over the cold winter

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months. The latter is also consistent with the need for energy conservation by greatly reducing ATP-expensive protein synthesis during diapause and/or in the frozen state. Making and then sustaining chaperones over the winter months is also consistent with the pattern of cryoprotectant polyol synthesis which likewise occurs in the autumn and is then sustained over the winter (Storey, 1990).

Acknowledgements This work was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada to K.B.S. and the Canada Research Chairs program.

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