1The relationship between thermal resistance and expression of inducible heat shock proteins, especially Hsp70, depends on the species and temperature treatments. The induction of Hsp70 has been shown to be essential for heat stress survival in a number of species, yet the maximum protein expression levels do not coincide with peak survival after heat hardening in Drosophila.
2Here we study the functional relationship between heat-induced expression of the heat shock protein Hsp70, and thermal resistance in adult Orchesella cincta by comparing thermal resistance (survival of 37·4 °C for 60 min) with Hsp70 gene and protein expression levels, all three measured at time points 2, 4, 6, 23, 27, 49 h after a heat hardening treatment (35·4 °C for 60 min).
3Thermotolerance increased over time after heat hardening until 49 h after exposure when the experiment ended. On the other hand the expression of hsp70 messenger RNA reached a peak within the first 2 h and then sharply decreased after 6 h. Within 23 h hsp70 expression was back to control levels.
4Surprisingly, protein levels of Hsp70 followed thermotolerance and reached the highest levels 49 h after heat hardening. A significant positive association was found between thermotolerance and Hsp70 protein levels, but not with hsp70 mRNA levels.
5Our results support a strong correlation between Hsp70 expression levels and thermal resistance following a heat hardening treatment. They also show that gene and protein expression follow different dynamics, a difference that may be important for our understanding of the role of candidate genes in functional studies.
Both the improved heat resistance and the negative fitness effects of heat hardening have often been associated with the induction of heat shock proteins (Dahlgaard et al. 1998; Krebs & Feder 1998a; Gregersen et al. 2001; Sørensen et al. 2003). Heat stress causes thermal denaturation of proteins, which in turn induces heat shock proteins. These proteins help prevent inappropriate protein aggregation in organisms by binding to the denaturated protein; they thus prevent its hydrophobic domains from associating with other damaged proteins (Feder & Hofmann 1999; Sørensen et al. 2003). Although under non-stressful conditions accumulated levels of heat shock proteins can interfere with regular protein function and constitute a fitness cost (Georgopoulos & Welch 1993; Sherman & Goldberg 2001), the heat shock response becomes increasingly important during stressed situations.
However, the function of specific heat shock proteins, and Hsp70 in particular, for inducible thermotolerance remains unclear. In certain organisms the levels of Hsp70 gene expression (Bettencourt et al. 2008) as well as the synthesis and persistence of higher molecular weight heat shock proteins correlate well with the acquired thermotolerance (Landry et al. 1982; Li & Werb 1982). Yet in other organisms this is not the case (Yocum & Denlinger 1992; Dahlgaard et al. 1998) and the relative importance of heat shock proteins for the development of thermotolerance in different species has therefore been questioned. Furthermore, the induction of heat shock proteins in response to heat hardening shows dependency on the environment. For example, individuals originating from hot environments express lower levels of heat shock proteins than individuals originating from warmer environments (Sarup et al. 2004; Sørensen et al. 2005b). Therefore, the heat shock response reflects the severity of the stress experienced by the organism, but also its evolutionary background (Köhler et al. 1999; Sørensen et al. 2001).
A number of studies have focused specifically on the relevance of Hsp70 in thermotolerance (Krebs & Feder 1998b; Sørensen et al. 2003). They have provided important information on the heat shock response and thermotolerance as seen from an ecological and evolutionary perspective. For example, studies using an extra copy strain of Drosophila melanogaster, which has an additional 6 hsp70 transgenes, have shown that these strains perform better under hot conditions than control strains although too high levels of Hsp70 can actually decrease thermotolerance (Krebs & Feder 1998b). This indicates that manipulation of a single trait (Hsp70 expression) is sufficient to alter thermotolerance, yet natural selection may balance benefits and costs of high and low expression levels in natural populations (Sørensen et al. 2003). Therefore we expect the specific patterns of the heat shock response to depend on the actual and past thermal conditions of the organism.
Soil organisms are exposed to quite different but well-defined and commonly more stable thermal conditions compared to other model organisms, such as D. melanogaster. Because of the winglessness of many soil organisms they usually have a reduced ability to escape unfavourable temperature conditions. Earlier studies have shown that soil organisms show local adaptation to temperature regimes (Bahrndorff et al. 2006; Liefting & Ellers 2008), and that there is variation in thermal responses (Bahrndorff et al. 2007; Driessen, Ellers & van Straalen 2007; Ellers et al. 2008). Unfortunately, hardly any studies address the heat hardening and the associated expression of heat shock proteins in soil ecosystems (but see Selvan et al. 1996). Here, we want to describe the heat hardening response in a soil arthropod, Orchesella cincta (Collembola) (Fig. 1). We look at the functional relationship between heat resistance and the induction of heat shock protein Hsp70 and Hsp70 gene expression at various intervals after heat hardening. Our aims are threefold; first, to establish if the heat hardening response in O. cincta is similar to above-ground insects such as D. melanogaster. We expect that soil-dwelling species such as O. cincta respond slower because they live in a thermally more stable environment. Second, we investigate how Hsp70 changes during heat hardening, both at the protein and mRNA level. Based on most other studies we expect that Hsp70, both RNA and protein, will increase shortly after the hardening treatment. The last aim is to relate changes in Hsp70 (both protein and RNA levels) to changes in heat resistance, as this seems to be dependent on the species in question.
origin and maintenance of populations
A mass population was formed by individuals collected from a site in the Netherlands (Roggebotzand: 52°34′N, 05°47′E). The population was founded by 300–400 individuals to obtain sufficient genetic material. Springtails were held in hard plastic containers containing a substrate of water-saturated plaster of Paris. The culture was kept at 16 ± 1 °C in a 12 : 12 h light : dark regime and fed on small twigs with algae growing on them. Twigs were frozen at −20 °C, 7 days prior being fed to the springtails to avoid biological contamination.
thermal exposure conditions
Heat hardening treatment
To induce thermotolerance, springtails were transferred to glass vials containing slightly moistened foam at the bottom and moistened foam stoppers and exposed to a heat hardening treatment (35·4 °C for 60 min in a waterbath). The heat hardening temperature chosen was based on a prior pilot experiment. After heat hardening treatment, springtails were transferred back to control conditions and allowed to recover for different lengths of time (2, 4, 6, 23, 27 and 49 h). Individuals having the longest recovery period were exposed first followed by the other groups so that all groups ended recovery time simultaneously. The control group was not exposed to a heat hardening treatment, but individuals were otherwise kept under the same condition at 16 °C. After the recovery, all experimental individuals were either frozen at −80 °C for future mRNA or protein analyses or assayed for thermotolerance. Ten individuals per vial and 10 vials per treatment were tested for thermotolerance and 10 individuals per vial and five vials per treatment were used for mRNA and protein measurements.
Thermal stress resistance
Springtails assayed for thermal resistance were exposed as above, but to a potentially lethal heat stress (37·4 °C) for 60 min. Ten individuals per vial and 10 vials per treatment were tested. After exposure individuals were allowed to recover for 24 h before mortality was assayed. Individuals that were able to walk in a coordinated fashion after gentle stimuli with a fine brush were considered as survivors.
For determination of Hsp70 protein expression levels each of five vials per treatment was homogenized individually and run on a SDS-page and subsequently analysed by Western-blotting. Springtails were homogenized in ice-cold phosphate-buffered saline (PBS) containing 2 mM PEFA block and 1% (volume) antiprotease cocktail (100 µL mL−1 pepstatin A, 50 µL mL−1 leupeptin, 10 mM benzamidine, 10 mM sodium metabisulfite). The homogenate was centrifuged for 30 min at 13 000 g at 4 °C. Subsequently each sample was aliquoted into two replicate samples and frozen at −80 °C. The total protein concentration in the supernatant was determined by means of a BCA assay (Pierce Biochemicals) according to manufacturer's instructions. Constant protein weights were analysed and aliquots of 100 µg for O. cincta were loaded in each lane for each treatment. To ensure that equal protein weights were loaded between treatments we incubated samples with monoclonal antibody for actin as well (Affinity BioReagents). These samples did not differ substantially in amount of actin and ensured that comparisons between-treatments and control comparisons could be made. Comparison of Hsp70 protein expression between the different treatments was corrected by incorporating the level of actin.
Protein of the total supernatant was separated by mini-gel SDS-page (10% acrylamide, 0·4% bisacrylamide, 1 h at 150 V). Two microlitres of MagiMark™ XP Western Standard (Invitrogen) was used as standard. Protein was transferred to nitrocellulose and the filter blocked in 5% skim-milk in PBS (137 mM NaCl, 2·7 mM KCl, 4·3 mM Na2HPO4, 1·47 mM KH2PO4, pH 7·1) for 1 h at room temperature. After washing in buffer for 2 × 5 min in 1% non-fat milk PBST, the primary antibody was added and incubated overnight at 4 °C (different incubation times did not affect expression patterns). Immunodetection was performed using two different primary antibodies. Monoclonal antibody (mouse anti-human hsp70; ABR, Affinity BioReagents, dilution 1 : 5000 in PBS) was used for detection of Hsp70 and another monoclonal antibody (mouse anti-Arabidopsis actin; ABR, Affinity BioReagents, dilution 1 : 1000 in PBS) was used for detection of β-actin. After repeated washing for 4 × 5 min in 1% non-fat milk PBST the nitrocellulose filter was incubated in secondary antibody goat anti-mouse IgG (H + L) coupled to peroxidase (Pierce, dilution 1 : 1000 in PBS) for 1 h at room temperature. The membrane was subsequently washed 6 × 5 min in 1% non-fat milk PBST as before. Signal was developed with a chemiluminescent system (Super Signal West Dura Extended Duration Substrate, Pierce) according to the manufacturer's instructions. For each treatment, five biological replicates were analysed for generalities in expression patterns. To enable us to compare controls and treated replicates these samples were always run on the same gel and with equal amounts of protein loaded onto each lane. Quantification of protein bands (Hsp70 and actin) on the blots was done using Versa Doc Imaging System (Biorad) and levels of Hsp70 were calculated relative to the actin level.
partial gene sequence of orchesella cincta hsp70 and design of qpcr primers
To obtain the (partial) sequence of the inducible Hsp70 gene of O. cincta we designed degenerate primers (Table 1) on the Hsp70 homologues of D. melanogaster (GenBank: AE014297) and Folsomia candida (Collembase.org: Fc_nor_51C12 and Fc_phe_02A11 (Timmermans et al. 2007)). PCR was performed using the GoTaq® Flexi DNA polymerase system of Promega. Cycling parameters were: 5 min 95 °C, 30 × (15 s 95 °C, 45 s 54 °C, 1 min 72 °C), 5 min 72 °C. The PCR product was cloned using the pGEM®-T Vector System of Promega in XL-1 Blue subcloning-grade competent cells of Stratagene. Plasmids were isolated from four positive clones and the inserts were sequenced with BigDye® Terminator v1·1 chemistry (Applied Biosystems) on an ABI PRISM 3100 Genetic Analyzer. The sequences were deposited in GenBank under the Accession numbers FJ009069–FJ009072 and the consensus sequence was used for further analysis. To confirm the identity of the consensus Hsp70 sequence, it was subjected to blastn and blastx (Altschul et al. 1990) comparison with the GenBank non-redundant nucleotide (nr/nt) and protein (nr) data base, respectively.
Table 1. Degenerate primers of the Hsp70 homologues of D. melanogaster and F. candida and RT-PCR primers developed on the consensus Hsp70 sequence
5′-TCC ATC AAC CCR GAY GAR GCW GT-3′
5′-CGW CCC TTG TCG TTY TTG ATS G-3′
5′-ATC AAA GTT TAT GAG GGA GAG CGA-3′
5′-AAG GTG ACT TCA ATT TGT GGC AC-3′
Quantitative Real Time PCR primers were developed on the consensus Hsp70 sequence using software package Primer Express version 1·5 (Applied Biosystems) to have an annealing temperature of 60 °C, and to amplify an amplicon of 80–120 bp with 45–55% GC content (Table 1).
In order to determine PCR efficiency, standard curves were obtained in triplicate for the RT-PCR primer set with fourfold dilutions of a standard batch cDNA (Pfaffl 2001). Quantitative RT-PCR was performed on a DNA engine Opticon (MJ Research), using universal cycling conditions (15 min at 95 °C; 15 s at 95 °C, 1 min 60 °C, 40 cycles) with a single fluorescence measurement and a melting curve program (60–90 °C with a heating rate at 0·1 °C s−1 and fluorescence measurement every second).
rna extraction and quantitative rt-pcr analysis
After the treatments animals were frozen in liquid nitrogen and stored at −80 °C. Animals (10 individuals per vial) were crushed and RNA was isolated using SV Total RNA isolation system (Promega). For cDNA synthesis 5 µL of total RNA (approximately 100 ng RNA µL−1) was reverse transcribed using 200 U MML-V reverse transcriptase (Promega) and 0·5 µg oligo(d)T, according to the manufacturer's instructions. The cDNA samples were 1 : 3 diluted and 2 µl was used in 20 µL PCR reaction volumes containing forward and reverse Hsp70-RT primers and Power SYBR Green PCR Master Mix (Applied Biosystems). Quantitative RT-PCR was performed in triplicate for each sample as described above. A mean normalized expression value (MNE) was calculated from the obtained Ct values with the Q-Gene module29 using β-actin as a reference gene for normalization of input cDNA.
Survival fractions were acrsin-square root transformed to prevent the variance to be a function of the mean and improve normality. Levene's test was used to test for equality of variances. Subsequently we used one-way anova to test for treatment effects on survival and Scheffe's post hoc test to test for differences between treatments. Welch test was used when assumptions of equality of variance was not met (expression of Hsp70) and Games–Howell post hoc test to test for differences between treatments. Due to no detectable levels of Hsp70 protein in some groups of treatments, a nonparametric Kruskal–Wallis test was used to test for treatment effects. Correlation analysis (Spearman correlation coefficient) was used to test for associations between thermotolerance and Hsp70 protein levels and mRNA expression. Analyses were performed using the statistical package spss 14·0 (SPSS Inc., Chicago, IL).
induction of thermal heat resistance after heat shock
In the control group (Fig. 2), which did not experience prior heat hardening, survival after heat shock was 31%. Survival gradually increased with time after hardening and reached a maximum in the 27 and 49 h group. One-way anova shows that time after hardening explains a significant proportion of survival (F6,69 = 10·65, P < 0·0001). Post hoc test revealed that survival of the control group without hardening was significantly lower compared to 23, 27 and 49 h after hardening (P < 0·05) and survival 2 and 4 h after hardening were significantly lower compared to 27 and 49 h past hardening (P < 0·05) (Fig. 2).
induction of hsp70 mrna expression after heat shock
The partial Hsp70 gene sequence obtained for O. cincta was 443 bp long and showed highly significant similarity to homologous Hsp70 nucleotide (Crassostrea gigas; AF144646; 7e-56; 76% nucleotide identity) and protein (D. melanogaster; AAW34354; 1e-59; 84% amino acid identity).
In the control group without heat hardening, expression of Hsp70 mRNA was low. However, expression of Hsp70 sharply increased and reached its peak after only 2 h of recovery (Fig. 3). At 4 and 6 h after hardening treatment expression was elevated but decreasing. After 23 and 27 h from hardening treatment expression levels were back to the control level. The Welch test shows that time after hardening explains a significant proportion of Hsp70 mRNA (F6,12·015 = 40·547, P < 0·001). Post hoc tests revealed that Hsp70 mRNA 2, 4 and 6 h after hardening treatment was significantly higher than in the control 23 and 27 h past hardening (P < 0·05). Furthermore, Hsp70 mRNA 2 h after hardening was significantly higher (P < 0·05) compared to all treatments except at 4 h.
induction of hsp70 protein levels after heat shock
Levels of protein Hsp70 were not detectable in the control treatment and 2 h after hardening treatment. However, levels of Hsp70 gradually increased thereafter (Fig. 4) and at 4 and 6 h after the hardening treatment expression was elevated but with large variation. The protein level continued to increase at 23 and 27 h after the hardening treatment and reached its peak at 49 h after hardening. A Kruskal–Wallis test revealed a significant effect of treatment (χ2 = 25·191, df = 6, P < 0·001).
the association between hsp70 and thermal tolerance
We performed correlation analyses to test if higher levels of Hsp70 (mRNA and protein) were associated with increased thermotolerance. The correlation coefficients revealed that there was a highly significant correlation between survival and Hsp70 protein levels (r = 0·918, n = 7, P < 0·01), but not for Hsp70 mRNA (r = −0·126, n = 7, P = 0·788).
Heat hardening treatment induces thermotolerance in a wide range of organisms. The induced heat tolerance persists for different lengths of time depending on the species being examined (Landry et al. 1982; Li & Werb 1982; Carretero et al. 1991; Dahlgaard et al. 1998). The present results show that a heat hardening treatment induces thermal resistance of O. cincta to temperatures which are otherwise lethal (Fig. 2). Protein levels of Hsp70 followed thermotolerance and reached the highest levels 49 h after heat hardening, whereas this was not the case for Hsp70 mRNA, highlighting that protein levels of Hsp70 are temporally associated with thermotolerance in O. cincta (Figs 3 and 4).
The response to the hardening treatment conformed to the general heat hardening pattern found in other organisms, although in O. cincta heat resistance was maintained for a relatively long period of time. Thermal resistance reached its peak after 27 h and remained elevated for at least 49 h, when the experiment ended (Fig. 2). This pattern was closely matched by the protein levels of Hsp70. Such a prolonged and stable response is not exceptional: in some fish species high levels of Hsp70 were maintained for at least 5 days after a 2-h heat-shock (Bierkens 2000). Also, in chironomids thermotolerance remained elevated until 72 h past hardening and Hsp70 protein expression remained elevated 12–34 h after the hardening treatment (Carretero et al. 1991). In contrast, in D. melanogaster and in mammalian cell cultures Hsp expression and heat resistance generally return to almost normal after 24–32 h (Dahlgaard et al. 1998; Feige et al. 1996). It should however be pointed out that the results are based on individuals from one population of O. cincta and recent results have shown genetic variation for plastic traits in this species (Liefting & Ellers 2008; Bahrndorff et al. 2008).
The rate of induced protein production after heat hardening in O. cincta is particularly slow: although elevated protein levels could be detected 4–6 h after heat hardening, protein expression levels still increased until the end of the experimental period (49 h) (Fig. 4). Hsp70 protein levels peak much faster in D. melanogaster, that is 2 h past hardening, which is even before any increases in thermotolerance are apparent (Dahlgaard et al. 1998). It is unlikely that the different rates of protein production are due to changes in the speed of mRNA Hsp70 expression. The temporal dynamics of mRNA Hsp70 levels found in the present study (Fig. 3) are in agreement with transcriptional profiling of heat-induced thermotolerance in D. melanogaster (Sørensen et al. 2005a), where mRNA levels of most stress proteins are almost back to control levels within 6–8 h after the heat hardening treatment. Rather, protein production seems exceptionally fast in D. melanogaster, as other taxa also show a delayed onset of protein production starting after 6 to12 h of recovery. For example, chironomids (C. thummi) reach maximum Hsp70 elevation 12 h after the hardening treatment (Carretero et al. 1991), bay scallops reach maximum Hsp levels after 6 h (Brun et al. 2008), and trout Hsp72 peak between 6 and 24 h after heat hardening (Werner et al. 1996).
A possible explanation for the striking discrepancy in temporal dynamics of protein levels and heat resistance between D. melanogaster and O. cincta can be found in the natural thermal regime experienced by the different species used. Under natural conditions, O. cincta lives on the soil surface, but it can retreat to deeper soil layers where the temperature is less variable and more predictable (Bahrndorff et al. 2008). The thermal regime of soil habitats is more buffered than the ambient temperatures experienced by most insects (Liefting & Ellers 2008). Based on the predictions of Krebs & Feder (1998b) individuals of O. cincta should therefore produce Hsp70 slower than D. melanogaster. Also, the temperature conditions during the recovery period can affect the speed of protein production. In Dahlgaard et al. (1998) Drosophila individuals were kept at 25 °C after the hardening treatment and in the present study individuals were kept at 16 °C, slowing the turnover rate of Hsp70 protein.
Another cause of variation in Hsp70 protein levels is the severity of the hardening treatment. In D. melanogaster cell lines recovery and synthesis of Hsp70 is proportional to the severity of the stress experienced (DiDomenico et al.1982). A similar relation between stress exposure and Hsp70 levels is found in the Collembolan O. bifasciata, when exposed to different concentrations of heavy metals. These soil invertebrates show low concentrations of Hsp70 levels after moderate exposure, while induced levels of Hsp70 increase with heavy metal exposure until reaching a non-compensation zone where Hsp70 levels decrease (Eckwert et al. 1997; Köhler et al. 2000). Hence, at high levels of metal exposure Hsp70 actually decreases which is likely to be due to pathological cell damage. Whether such an effect could explain the present results is uncertain.
The association between inducible thermotolerance and Hsp70 protein levels has been described in a number of studies using different organisms, exposures and time scales. Some studies have found a strong relation between thermal tolerance and Hsp70 protein expression (Carretero et al. 1991; Eckwert et al. 1997; Landry et al. 1982; Li & Werb 1982), whereas other studies have not (Dahlgaard et al. 1998; Krebs et al. 1998b; Yocum et al. 1992). The complexity is exemplified in a study by Krebs & Feder (1998b) comparing a control and extra-copy lines of D. melanogaster, where a tight association was found between inducible thermotolerance and Hsp70 levels until critically high levels of Hsp70 were reached. The correlation not only illustrates the role of Hsp's in thermotolerance at moderate stress levels, but also highlights that expression of Hsp70 comes at a cost (Sørensen et al. 2003). Furthermore, Hsp70 expression is eliminated when stress is absent (Solomon et al. 1991) and also after the recovery phase has ended (DiDomenico et al. 1982). As pointed out by Krebs & Feder (1998b) variation in Hsp70 has positive and negative effects and the trade-offs between them can affect regulatory control. The benefits and costs of different regulatory patterns may vary with different thermal environments. The temporal dynamics of Hsp70 expression levels following heat hardening in the present study also raises a number of questions concerning costs and benefits of heat hardening in soil organisms. Contrary to results obtained on Drosophila Hsp70 levels do not seem to disappear fast in O. cincta. This obviously indicates a different temporal dynamics of cost and benefit functions between these organisms.
The present results highlight that we need to increase our understanding of the heat hardening response. Transcriptomic and metabolomic tools have yielded valuable information on the heat hardening response in fruit flies (Sørensen et al. 2005a; Malmendal et al. 2006). However, we need to broaden the use of model systems to include species from different thermal niches and with different patterns of hardening responses and the underlying cost : benefit ratios. At present we do not have a full understanding of why some species show a similar hardening response whereas others do not.
SB was supported by a grant from the graduate school ISOBIS and Natural History Museum of Aarhus and an exchange grant from the European Science Foundation (ESF). JE was supported by the Netherlands Organization for Scientific Research, VIDI-grant 864·03 003. This study was further supported by grants from the Danish Natural Science Research Council (VL).