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Developmental upregulation of inducible hsp70 transcripts, but not
the cognate form, during pupal diapause in the flesh fly,
Sarcophaga crassipalpis
Joseph P. Rinehart, George D. Yocum, David L. Denlinger
*Department of Entomology, The Ohio State University, 103 Botany and Zoology Building, 1735 Neil Avenue, Columbus, OH 43210, USA
Received 23 June 1999; received in revised form 28 December 1999; accepted 25 January 2000
Abstract
Partial clones of the Sarcophaga crassipalpis heat shock protein 70 (hsp70) and of heat shock cognate 70 (hsc70) were developed by RT-PCR and library screening respectively. These clones were used to probe total RNA northern blots for the expression of transcripts in response to high and low temperature stress and in conjunction with the entry into an overwintering pupal diapause. In nondiapausing individuals, hsp70 was highly expressed in response to a 40°C heat shock, while hsc70 was unaffected by the heat stress. In contrast, both hsp70 and hsc70 were upregulated in nondiapausing flies following a210°C cold shock. In diapausing pupae, hsp70 was highly upregulated during diapause, even at a non-stress temperature of 20°C. Upregulation was initiated at the onset of diapause and persisted throughout diapause. During diapause, heat shock did not further elevate the level of hsp70 expression. Within 12 h after diapause was terminated, hsp70 ceased to be expressed. The expression of hsc70 was unaltered by diapause. The developmental regulation of hsp70 in relation to diapause suggests a critical role for this stress protein during insect dormancy.2000 Elsevier Science Ltd. All rights reserved.
Keywords: Diapause; hsp70; hsc70; Heat shock; Cold shock
1. Introduction
To survive seasonally reoccurring, chronic forms of environmental stress, such as cold or dry seasons, most insects enter the dormant state of diapause. Initiated by environmental cues, diapause is characterized by devel-opmental arrest, decreased metabolism (Tauber et al., 1986), an increase in resistance to stresses including increased cold hardiness (Adedokun and Denlinger, 1984; Lee and Denlinger, 1985), and the activation of a specific set of genes (Flannagan et al., 1998). Diapause has been well defined in several species, including the flesh fly Sarcophaga crassipalpis. This temperate spec-ies responds to short daylength and cool temperatures by entering diapause early in the pupal stage, where it remains for several months until favorable conditions return (Denlinger, 1972).
* Corresponding author. Tel.:+1-614-292-8209; fax: +1-614-292-7865.
E-mail address: [email protected] (D.L. Denlinger).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 2 1 - 7
For protection against more acute forms of environ-mental stress, insects rely in part on the well described heat shock proteins (hsps). These proteins are rapidly upregulated in response to such environmental insults as temperature extremes, anoxia, and various chemical con-taminants (Linquist, 1986; Feder, 1996). In S. crassi-palpis, the dominant hsp expressed in response to ther-mal stress is a member of the hsp70 family (Joplin and Denlinger, 1990).
diapause, in the absence of temperature stress (Yocum et al., 1998). How these proteins may function in the long-term developmental arrest associated with diapause is unclear. The suggestion that hsps may persist for long periods during diapause (Yocum et al., 1998) is especially intriguing because extended expression of hsps can lead to detrimental effects at the cellular level (Feder et al., 1992). This prompted us to investigate the potential contribution of hsp70, the dominant hsp of S. crassipalpis, to diapause. Are hsp transcripts present throughout diapause or is their expression pattern in response to stress altered during this state? To answer this question, we have used partial clones of inducible and cognate members of the hsp70 family in S. crassi-palpis to observe their expression patterns after acute stress and during diapause. We show that when the fly is not in diapause, transcripts of these proteins exhibit the expected pattern of upregulation during thermal stress. Of greater interest, we observed that hsp70 tran-scripts are upregulated as the fly enters pupal diapause, even in the absence of an environmental stressor, and high temperature stress during diapause has no further effect on transcript levels. Thus, hsp70 expression is a developmentally regulated component of the diapause program in S. crassipalpis and may be involved in the protection inherent to the diapause state.
2. Materials and methods
2.1. Insect rearing
All flies were obtained from an established laboratory colony of the flesh fly Sarcophaga crassipalpis Mac-quart. The colony of nondiapausing flies was reared under long day conditions (15:9; light:dark) at 25°C while diapausing pupae were obtained by raising the par-ental generation under short day conditions (12:12; light:dark) at 25°C and the larvae at 12:12 L:D at 20°C, as previously described (Denlinger, 1972). When direct comparisons were made between nondiapausing and diapausing flies, both groups were raised as larvae at 20°C while maintaining their respective light regimes. Diapause was terminated by application of 5 µl hexane directly to the heads of diapausing individuals as for-merly documented (Denlinger et al., 1980).
2.2. Temperature treatments
Fifteen flies were placed in thin-walled 13×100 mm Pyrex test tubes plugged with cotton. Temperature alter-ations were achieved by submersing the tubes in a Lauda model RM20 glycerol bath.
2.3. Clone development
The partial clone of S. crassipalpis hsp70 was developed by PCR, using primers that annealed near the 39end of the transcript. The resulting clone was 428 bp, and corresponds to bases 1403 through 1840 of the D. melanogaster hsp70 open reading frame. Our clone is 64% identical to D. melanogaster at the nucleotide level and 87% identical at the amino acid level. The clone of S. crassipalpis hsc70 was isolated as a result of a flesh fly library screen for differentially expressed genes asso-ciated with diapause (Flannagan et al., 1998). This clone was identified as one whose expression was not affected by diapause. It is 885 bp in size, corresponding to bases 1376 through the end of the open reading frame of the D. melanogaster transcript. It exhibits 85% identity to the D. melanogaster hsc70-4 gene at the nucleotide level and 92% identity at the amino acid level. Nucleotide sequences for both clones were deposited in Genbank, with accession numbers AF107338 for hsp70 and AF107339 for hsc70.
2.4. RNA isolation and Northern blot hybridization
Total RNA was isolated from whole flies or specific tissues by homogenization in TRIzol reagent (GibcoBRL) using standard protocol. Samples from three flies were pooled for each time point, with 20 µg RNA from each pooled sample loaded on a 1.5% aga-rose, 0.41 M formaldehyde gel for separation by electro-phoresis. Samples were transferred to a Magnacharge+ nylon membrane (Micron Separations, Inc.) by down-ward capillary action using alkaline transfer buffer (Schleicher and Schull, Inc.).
Northern blot hybridization was then performed as previously described (Sambrook et al., 1989) using our hsp70, hsc70 and 28s ribosomal RNA control clones as templates to make DNA probes. Each probe was labeled with 32
3. Results
3.1. Expression of hsp70 and hsc70 during heat shock
Transcript levels of hsp70 in nondiapausing pharate adults increased dramatically within 15 min after the onset of heat shock at both 40°C and 45°C (Fig. 1a). While the rapidity of response did not differ between the two treatment temperatures, differences in the levels of response were evident on equally loaded gels (as determ-ined by 28s rRNA probe binding). Neither heat shock temperature nor length of exposure affected the duration of hsp70 expression; in all cases the levels were signifi-cantly reduced 2 h after treatment (Fig. 1b). The expression levels of hsc70 did not substantially change as a result of heat shock (Fig. 1a). Although it has pre-viously been documented that different life stages of S. crassipalpis have different degrees of tolerance to heat (Chen et al., 1991), no differences in hsp70 expression in response to high temperature were noted among wan-dering larvae, true pupae, pharate adults, and 1 day old adults (data not shown).
Fig. 1. Expression of (A) hsp70 and hsc70 in nondiapausing pharate adults as a result of different durations of heat shock at 40°C and 45°C and (B) the decline of hsp70 expression at hourly intervals after heat shock at 40°C and 45°C. Flies were held at 25°C following the heat shock. 28s was used as the control gene.
3.2. Expression of hsp70 and hsc70 during cold shock
Fig. 2. Expression of hsp70 and hsc70 at hourly intervals after 15, 30, or 60 min exposures to210°C, with 28s binding used as a control.
The expression of hsc70 was altered by cold shock as well. While exposure to 0°C did not cause an increase in hsc70 transcript levels (data not shown), an exposure to210°C initiated upregulation, with little response seen after a 15 min exposure and a larger response after a 60 min exposure (Fig. 2). As in the case of hsp70 expression, upregulation did not occur during the cold shock itself, rather a recovery period was required for transcripts to be upregulated, with levels appearing 50% higher by 1 h post treatment (as determined by band average gray scale densities) and falling to 40% above baseline by 4 h post treatment.
3.3. Expression of hsp70 during diapause
At 20°C, a non-stress temperature, hsp70 was expressed during pupal diapause. Although diapause-destined wandering larvae did not express hsp70, levels were easily detected in early (15 d), mid (25 d), and late
(55 d) stages of pupal diapause (Fig. 3a). By contrast, nondiapausing individuals showed no such increase. When diapause was terminated with a topical application of hexane, hsp70 was rapidly downregulated, dropping below detectable levels 12 h after hexane application (Fig. 3b). Transcripts of hsc70 showed no difference in levels throughout the course of diapause (Fig. 3a) and remained the same in diapausing and nondiapausing individuals. The expression of hsp70 was not uniform in different tissues. On an equally loaded northern blot (Fig. 3C) the highest expression was noted in the brain, followed by the epithelium (95% of brain), gut (84% of brain) and fat body (53% of brain). Interestingly, both hsp23 and hsc70 showed similar tissue patterns of expression (data not shown).
3.4. Expression of hsp70 and hsc70 during temperature stress and diapause
During diapause, environmental stress in the form of a 1 h exposure to 40°C did not further elevate the levels of hsp70 transcripts as determined by northern blot (Fig. 4a), a result that contrasts significantly with that seen in nondiapausing individuals (Fig. 1). Levels of hsc70 transcripts were affected by thermal stress during diapause, with upregulation especially evident upon recovery from cold shock (Fig. 4b).
4. Conclusions
The expression of both hsp70 and hsc70 during heat shock in nondiapausing individuals of S. crassipalpis is similar to what is seen in other models; while hsc70 lev-els were not affected by the higher temperatures, hsp70 was rapidly upregulated. Of additional interest is the fact that the rapidity of the hsp70 response did not differ at the two high temperatures tested (40°C and 45°C), but the level of expression was affected. This is also true for hsp23 expression in S. crassipalpis (unpublished results). Hsp70 protein profiles indicate similar results in our model (Joplin and Denlinger, 1990), as well as in the gypsy moth Lymantria dispar (Yocum et al., 1991), the brown apple moth Epiphyas postvittana (Lester and Greenwood, 1997), and others. Additionally, our tran-script data confirms protein data for S. crassipalpis
Fig. 4. Expression of hsp70 and hsc70 in diapausing pupae upon recovery from (A) a 1 h heat shock at 40°C and (B) a 1 h cold shock at 0°C. Pupae used in this experiment had been in diapause for 30 days (mid diapause). nl= normal, untreated controls, 28s binding presented as a control.
which shows a rapid return to normal levels following the return to ambient temperature (Yocum and Denlinger, 1992).
The expression patterns after cold shock in nondi-apausing individuals also agree with the protein patterns previously identified; while an exposure to 0°C does not cause hsp70 expression, a210°C exposure does (Joplin and Denlinger 1990). Additionally, the expression is not seen immediately after cold treatment, rather the animals must recover at room temperature before the increased levels are realized. Once upregulated, the levels appear to remain stable for a period of several hours. Cold shock also prompts the upregulation of hsc70, a response simi-lar to the upregulation of hsc70 reported in spinach and other plants in response to low temperature (Anderson et al., 1994; Neven et al., 1992). This data suggests that some members of the hsp70 family function in a funda-mentally different manner during high and low tempera-ture stress. The fact that a recovery period is needed before hsp70 expression and hsc70 upregulation is real-ized indicates that these genes are not involved in the immediate response to cold shock that leads to rapid cold hardening (Chen et al., 1987; Lee et al., 1987).
Most interestingly, our data indicates that hsp70 tran-scripts are regulated as a function of diapause, turning on as the animal enters pupal diapause, and turning off by 12 h after diapause termination. This data is in agree-ment with that shown for hsp23 in S. crassipalpis (Yocum et al., 1998), thus suggesting that at least two of the major heat shock proteins in this fly are upregul-ated during diapause.
The function of hsps during dormancy remains unclear. Recent data from our laboratory indicates that reduced cellular growth in the form of cell cycle arrest plays a critical role in the pupal diapause of S. crassi-palpis (Tammariello and Denlinger, 1998). Other researchers have suggested a role for hsps in cell cycle regulation; cultured Drosophila cells that have continu-ous expression of hsp70 exhibit a reduced rate of cellular growth (Feder et al., 1992), and Drosophila larvae that contain extra copies of hsp70 exhibit decreased growth and development (Krebs and Feder, 1997). Additionally, hsps have been implicated in programmed cell death in Caenorhabditis elegans (Madi et al., 1997). If the majority of negative effects resulting from hsp expression involve the inhibition of cellular growth and differentiation, this should not impair an organism in diapause and may actually aid in maintaining the diapause state. However, the connection between hsp70 expression and cell cycle control is mainly circumstan-tial and has yet to be tied to a specific cellular function of the hsp70 protein.
role for hsps during diapause is further supported by the fact that hsp70 (this study) and hsp23 (Yocum et al., 1998) transcript levels are not increased by high tem-perature stress when the animal is in diapause, and by data from gypsy moth diapause (Yocum et al., 1991; Denlinger et al., 1992). Gypsy moth diapause differs from that of the flesh fly in that the initiation of diapause and the establishment of cold hardiness are not directly linked. Gypsy moths enter an obligate diapause, but cold hardiness is not attained until low temperatures have been experienced. This is mirrored by the hsp70 expression pattern: the protein does not appear at the onset of diapause but only after the gypsy moth has experienced low temperatures (Yocum et al., 1991; Denlinger et al., 1992). In contrast, cold hardiness and diapause are directly linked in flesh flies, as is the expression of hsp70. When the fly enters diapause, it is cold hardy and expresses hsp70. When diapause is terminated, cold hardiness is lost and expression of hsp70 ceases. This strong correlation suggests a possible connection between the prolonged expression of hsp70 during diapause and the increased level of protection seen during this stage.
Recent data suggests that other models of dormancy may involve developmental regulation of hsps as well. In the nematode C. elegans, hsp90 transcript levels increase during the dauer dormant stage (Dalley and Golomb, 1992), and conidiospores of Neurospora crassa express transcripts for three high molecular weight stress pro-teins while in dormancy (Plesofsky-Vig and Brambl, 1985). Additionally, a small heat shock protein is upreg-ulated during embryonic diapause in the brine shrimp Artemia franciscana (Clegg et al., 1996).
This mounting body of evidence suggests a significant role for the continual expression of hsps during periods of dormancy. Deciphering how this expression is mediated and the function of these proteins during diapause may provide vital information towards under-standing the control of diapause at the cellular and mol-ecular levels.
Acknowledgements
This research was supported in part by NSF grant IBN-9728573 and USDA-NRI grant 98-35302-6659.
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