All tissues of larval Drosophila melanogaster express Hsp70, the major heat-shock protein of this species, after both mild (36 °C) and severe (38.5 °C) heat shock. We used Hsp70-specific immunofluorescence to compare the rate and intensity of Hsp70 expression in various tissues after these two heat-shock treatments, and to compare this with related differences in the intensity of Trypan Blue staining shown by the tissues. Trypan Blue is a marker of tissue damage. Hsp70 was rarely detectable before heat shock. Brain, salivary glands, imaginal disks and hindgut expressed Hsp70 within the first hour of heat shock, whereas gut tissues, fat body and Malpighian tubules did not express Hsp70 until 4–21 h after heat shock. Differences in Hsp70 expression between tissues were more pronounced at the higher heat-shock temperature. Tissues that expressed Hsp70 slowly stained most intensely with Trypan Blue. Gut stained especially intensely, which suggests that its sensitivity to heat shock may limit larval thermotolerance. These patterns further suggest that some cells respond primarily to damage caused by heat shock rather than to elevated temperature per se and/or that Hsp70 expression is itself damaged by heat and requires time for recovery in some tissues.

In response to heat and other stresses, nearly all organisms express heat-shock proteins (Hsps), which promote stress tolerance by functioning as molecular chaperones (Lindquist, 1993). Recent years have witnessed enormous progress both in the elucidation of chaperone function at the biochemical level and in the demonstration that heat-shock proteins are responsible for a large component of organismal thermotolerance (Morimoto et al. 1994). Progress has not been as rapid, however, in establishing how the activities of Hsps at the cellular level enhance the thermotolerance of the individual (Hartl, 1996). At a more descriptive level, the way in which tissue-specific expression of Hsps is temporally and quantitatively related to the thermotolerance of the various tissues and the patterns of cell damage that ensue during and after heat shock are poorly understood. Accordingly, we have examined tissue-specific patterns of Hsp70 expression and cell damage in Drosophila melanogaster, the fruit fly. In D. melanogaster, Hsp70 is the primary inducible heat-shock protein (Lindquist, 1981) and is the product of 10–12 nearly identical genes at the 87A and 87C loci (Ish-Horowicz et al. 1979a,b). This protein is not expressed before stress and is very tightly autoregulated as Hsp70 concentrations increase during stress and/or recovery from stress (Lindquist, 1993).

Although Hsps are best known for their inducibility by heat, the presence of non-native proteins within cells is sufficient to induce their expression (Parsell and Lindquist, 1994). Hsp70 expression, which can be detected immunologically, may therefore be symptomatic both of a direct response to high temperatures and of damage to cells and tissues by high-temperature stress (Hofmann and Somero, 1995). To examine tissue damage directly, we stained with Trypan Blue, a dye that is excluded from intact cells but is rapidly absorbed by dead or dying cells. Hsp70 expression and Trypan Blue staining may pinpoint thermosensitive regions within an organism. We therefore examined the order in which tissues first expressed Hsp70 during or after heat shock, and related this to Trypan Blue staining. We performed this experiment in larvae, the life stage of D. melanogaster most likely to experience lethal heat stress in nature (Feder, 1996; Feder et al. 1997).

Much of the extensive literature on Hsps in D. melanogaster and other insects has little bearing on the tissue specificity of Hsp expression and heat damage because it combines results for several different Hsps and/or tissues without distinguishing among them. Studies of whole D. melanogaster and cells in culture, which have been the most frequently used subjects of previous work, are not relevant to tissue-specific variation. All or most individual tissues of D. melanogaster (Tissiéres et al. 1974; Mitchell et al. 1979) and other dipterans (Nath and Lakhotia, 1989; Joplin and Denlinger, 1990; Tiwari et al. 1995) clearly increase their expression of unspecified members of the major families of Hsps upon heat shock. Seldom, however, have the individual Hsps been identified [but see Palter et al. (1986) and Singh and Lakhotia (1995)] or have the tissues under study been systematically exposed to heat shocks of graded severity.

We analyzed Hsp70 expression and tissue damage in the Chromosome II excision strain of Drosophila melanogaster (Welte et al. 1993). This strain, which was a control in some previous analyses of the effects of hsp70 copy number on thermotolerance, growth and Hsp70 expression (e.g. Krebs and Feder, 1997), contains a second-chromosome P-element insertion but expresses Hsp70 normally. Eggs were collected from, and larvae reared on, yeast–cornmeal–molasses–agar medium sprinkled with live yeast. Third-instar larvae (6–7 days post-laying) were separated from the medium in 3 mol l−1 NaCl (Ashburner, 1989) and transferred to 5 cm Petri dishes containing medium. This procedure does not affect Hsp70 concentration.

To characterize the thermal sensitivity of Hsp70 expression in entire larvae, third-instar larvae were exposed to constant temperatures between 33 and 40 °C for 1 h, followed by 1 h at 25 °C, as a recovery period. Other larvae were treated at 38.5 °C for 1 h, and then placed at 25 °C for variable periods. Whole-body Hsp70 concentration was then determined by enzyme-linked immunosorbent assay (ELISA) and is presented relative to a standard concentration, that produced by Drosophila S2 cells treated for 1 h at 36.5 °C and 1 h at 25 °C (Welte et al. 1993; Feder et al. 1996).

Fluorescent staining

Larvae were immersed in PBS (phosphate-buffered saline), and cuticle and muscle tissue were peeled from the body cavity. Larvae were then placed in 4 g l−1 paraformaldehyde (Fisher, T-353) in PBS (pH 7.3) and rotated for 30 min at room temperature (23 °C), rinsed three times with PBS, washed with rotation in PBST [5 g l−1 bovine serum albumin (Sigma A-6793), 5 g l−1 Triton X-100 (Sigma T-6878) in 1× PBS] for 30 min to permeabilize cells, and stored overnight at 4 °C in fresh PBST.

Tissues were incubated for 1 h in 1:1000 anti-Hsp70 rat monoclonal antibody, 7FB, which is specific for the Drosophila melanogaster heat-inducible Hsp70 family member (Velazquez and Lindquist, 1984). The secondary antibody was FITC-conjugated goat anti-rat IgG, affinity-purified F(ab′)2 fragments (Cappel 55747) prepared according to the instructions of the manufacturer and diluted 1:300 in PBST. To remove any nonspecifically binding components of the secondary antibody, each 1 ml of this dilution was incubated at room temperature for 1 h with 3–5 heat-shocked larvae that had not been treated with primary antibody. Then, Hoechst 33258 dye was added (10 μl ml−1 of a 1 mg ml−1 stock solution to secondary antibody) for coincident staining of nuclei. Samples were incubated with secondary antibody for 1 h, after which they were rinsed three times with PBS and washed for 30 min in PBST after addition of each antibody.

Tissues were transferred to a glass slide and mounted in several drops of 1 mg ml−1p-phenylenediamine in 70 % glycerol. Slides were examined with a Zeiss fluorescent microscope and photographed (2.5× camera lens) with 100 ASA Ektachrome film, using exposures of 5 s with a 10× objective and 40 s with a 4× objective. Comparisons of expression levels were possible for photographs taken with the same objective. All slides were scanned as Adobe Photoshop documents (Adobe Systems, Inc.), with assembly and text additions in PowerPoint (Microsoft Corp.).

Trypan Blue staining

Larvae were dissected as for Hsp70 analysis, a step requiring less than 10 min, immersed in 0.2 mg ml−1 Trypan Blue in PBS, and rotated for 30 min at room temperature to bring internal tissues into contact with dye. Groups of three larvae were then rinsed three times in PBS, washed for 30 min in PBS, and each group was immediately scored for Trypan Blue staining of tissues and cells. Scoring for these groups of larvae was based on an average composite index per larva: no color, 0; any blue, 1; darkly stained nuclei, 2; large patches of darkly stained cells, 3; or complete staining of most cells in the tissue, 4. As these data are sequential categories, differences due to treatment effects (control versus heat shock) and due to recovery time (immediately after heat shock versus 21 h after heat shock) were tested by Mann–Whitney U-tests. For presentation, images were taken with tissues in PBS and recorded on a Wild microscope with a direct computer feed. Staining for Hsp70 and Trypan Blue was not possible in the same larva, as Trypan Blue leaches from the sample after fixation.

In whole larvae, Hsp70 was undetectable in the absence of stress. Hsp70 level varies with temperature in third-instar larvae (Fig. 1A, F7,32=28.6, P<0.001), and the concentration of Hsp70 was greater after a 1 h exposure to 36 °C than at any other temperature tested. After 1 h of heat shock at temperatures lower than 36 °C, the concentration of Hsp70 declined rapidly (R. Krebs, unpublished results). In contrast, a 1 h exposure to 38.5 °C caused the concentration of Hsp70 to rise for many hours afterwards (Fig. 1B). The level of Hsp70 4 h after exposure to 38.5 °C for 1 h was significantly greater than the level immediately after the heat shock, and that after 21 h was significantly higher than that after 4 h (Tukey’s multiple-comparisons test, P<0.05).

Fig. 1.

Expression of Hsp70 in whole-body lysates of Drosophila melanogaster larvae relative to a standard, the concentration in Drosophila S2 cells after 1 h at 36.5 °C and 1 h at 25 °C. (A) Hsp70 level determined by ELISA after 1 h at the indicated temperature followed by 1 h at 25 °C; (B) Hsp70 concentration after 1 h at 38.5 °C and at the indicated time at 25 °C. Values are means ± S.E.M.; values of N are given beside the data points.

Fig. 1.

Expression of Hsp70 in whole-body lysates of Drosophila melanogaster larvae relative to a standard, the concentration in Drosophila S2 cells after 1 h at 36.5 °C and 1 h at 25 °C. (A) Hsp70 level determined by ELISA after 1 h at the indicated temperature followed by 1 h at 25 °C; (B) Hsp70 concentration after 1 h at 38.5 °C and at the indicated time at 25 °C. Values are means ± S.E.M.; values of N are given beside the data points.

In the immunohistochemical studies, autofluorescence was limited to a few tissues; for example, at the junction of the midgut and hindgut, and in parts of the trachea. Incubation with secondary antibody alone did not increase fluorescence. Non-heat-shocked samples incubated with both primary and secondary antibodies exhibited repeatable staining of a few cells in the base of the right and left lobes of the brain. Staining in these cells was never observed in the absence of primary antibody. Male gonadal disks occasionally fluoresced in the absence of heat treatment, and once quite strikingly, but this fluorescence was inconsistent.

All tissues expressed Hsp70 after 1 h at 36 °C, but differed in the intensity of their fluorescent signal (Fig. 2). Larval brain, salivary glands and imaginal disks stained most strongly immediately after stress (Fig. 2A), with the hindgut also showing a pronounced signal (Fig. 2C). When larvae were allowed to recover at 25 °C for 4 h after exposure to 36 °C for 1 h, the tissues that first showed high Hsp70 levels declined markedly in fluorescence, while staining in the gut was uniformly high (Fig. 2B,D). One exception was the Malpighian tubules, in which staining was initially low but became more pronounced than that of any other tissue by 4 h after exposure to 36 °C (Fig. 2D). By 24 h after exposure to 36 °C, Hsp70 was undetectable in all tissues, except faintly in the anterior midgut (not shown).

Fig. 2.

Hsp70 expression in larval tissues after a 1 h exposure to 36 °C and either (A,C) no recovery or (B,D) 4 h of recovery at 25 °C. A and B are for anterior tissues; C and D are for posterior tissues: sg, salivary glands; fb, fat body; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. At the presented level of photographic exposure, images of individuals at 25 °C are indistinguishable from background, representing undetectable expression in all tissues. Scale bars, 500 μm.

Fig. 2.

Hsp70 expression in larval tissues after a 1 h exposure to 36 °C and either (A,C) no recovery or (B,D) 4 h of recovery at 25 °C. A and B are for anterior tissues; C and D are for posterior tissues: sg, salivary glands; fb, fat body; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. At the presented level of photographic exposure, images of individuals at 25 °C are indistinguishable from background, representing undetectable expression in all tissues. Scale bars, 500 μm.

Immediately after a 38.5 °C heat shock, brain, salivary glands, imaginal disks and hindgut (the same four tissues showing pronounced staining at 36 °C) stained more intensely than all other tissues (Fig. 3), although the signal was relatively less than that obtained at 36 °C (Fig. 2). Almost no fluorescence was evident elsewhere. Four hours after the stress treatment, fluorescence levels increased in most tissues (Fig. 4) and tended to localize to nuclear regions. Fluorescence was patchy throughout the gut, and the many small regions of intense fluorescence (Fig. 4C) appeared to be from expression in the tiny tracheal cells responsible for aerating the broad cells of the gut proper. Alternatively, some of this fluorescence may have been in gut imaginal cells. Of all gut tissues, only in the gastric caeca was cytoplasmic fluorescence evident by 4 h after heat shock.

Fig. 3.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and no recovery at 25 °C. (A) Anterior tissues, (B) posterior tissues and (C) middle midgut. sg, salivary glands; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules; fb, fat body; fgd, female gonadal disk. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm.

Fig. 3.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and no recovery at 25 °C. (A) Anterior tissues, (B) posterior tissues and (C) middle midgut. sg, salivary glands; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules; fb, fat body; fgd, female gonadal disk. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm.

Fig. 4.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and 4 h of recovery at 25 °C. (A) Anterior tissues, (B) posterior tissues and (C) middle midgut. sg, salivary glands; fb, fat body; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm.

Fig. 4.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and 4 h of recovery at 25 °C. (A) Anterior tissues, (B) posterior tissues and (C) middle midgut. sg, salivary glands; fb, fat body; id, imaginal disks, gc, gastric caeca; pv, proventriculus; br, brain; mg, midgut; hg, hindgut; mt, Malpighian tubules. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm.

By 21 h of recovery, tissues that stained immediately after the 38.5 °C heat shock were much less fluorescent than gut and related tissues, especially caeca (Fig. 5). In contrast, gut tissues, Malpighian tubules and fat body now stained intensely. Fat body, which makes up a substantial proportion of the body mass in late-instar larvae, fluoresced less intensely than most other tissues after all other treatment conditions. Consequently, fat body fluorescence more closely paralleled Hsp70 levels in whole-body lysates than did fluorescence in other tissues. This general pattern, signal decrease in some tissues with signal increase in others, is dramatically evident from the faint patch in Fig. 5D, where the outline of a non-fluorescent female gonadal disk is clearly discernible amidst fluorescent fat body.

Fig. 5.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and 21 h of recovery at 25 °C (A) Anterior tissues, (B) posterior tissues, (C) middle midgut and (D) a region of the fat body surrounding the female gonadal disk. fb, fat body; gc, gastric caeca; mg, midgut; hg, hindgut; mt, Malpighian tubules; fgd, female gonadal disk. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm in A,B,C; 250 μm in D.

Fig. 5.

Hsp70 expression in larval tissues after 1 h of exposure to 38.5 °C and 21 h of recovery at 25 °C (A) Anterior tissues, (B) posterior tissues, (C) middle midgut and (D) a region of the fat body surrounding the female gonadal disk. fb, fat body; gc, gastric caeca; mg, midgut; hg, hindgut; mt, Malpighian tubules; fgd, female gonadal disk. Expression is identified by fluorescent staining with anti-Hsp70 monoclonal antibody. Scale bars, 500 μm in A,B,C; 250 μm in D.

Control larvae, as well as those receiving temperature treatments, stained with Trypan Blue in the anterior and posterior remnants of the muscle tissue following dissection, but otherwise showed minimal staining. This is consistent with tissue or cell damage in the region of dissection, but little elsewhere. In the control preparation depicted in Fig. 6A, gut staining, including caeca, upper midgut and lower gut (the region labeled hindgut, of which scoring may include damage in the most posterior part of the midgut) was uncharacteristically intense by comparison with other controls. Nevertheless, even in this preparation, the level of staining was much lower than in treated individuals (Fig. 6B,C). Gut stained slightly in the controls, probably due to damage from inadvertent stretching during dissection, but few other cell types stained with Trypan Blue in larvae not exposed to high temperature.

Fig. 6.

Trypan Blue staining in (A) an untreated larva, (B) a larva exposed to 38.5 °C for 1 h, and (C) a larva exposed to 38.5 °C for 1 h and then maintained at 25 °C for 21 h: fb, fat body; pv, proventriculus, gc, gastric caeca; mg, midgut; hg, hindgut. Larvae were dissected, immersed in Trypan Blue, which is excluded from healthy cells, and washed thoroughly. Thus, blue indicates tissue damage. Scale bar, 1 mm.

Fig. 6.

Trypan Blue staining in (A) an untreated larva, (B) a larva exposed to 38.5 °C for 1 h, and (C) a larva exposed to 38.5 °C for 1 h and then maintained at 25 °C for 21 h: fb, fat body; pv, proventriculus, gc, gastric caeca; mg, midgut; hg, hindgut. Larvae were dissected, immersed in Trypan Blue, which is excluded from healthy cells, and washed thoroughly. Thus, blue indicates tissue damage. Scale bar, 1 mm.

In comparison with controls, tissues of heat-shocked larvae (1 h at 38.5 °C) were more extensively stained with Trypan Blue, and staining varied among tissues (Fig. 7). Changes from control levels were relatively larger in caeca, midgut, brain and salivary glands, where differences were statistically significant (Mann–Whitney U-tests, P<0.05). The intensity of staining, however, was strongest in gut tissues (caeca, midgut and hindgut), but these tissues also showed more staining in controls than non-gut tissues (Fig. 7). Very little staining after heat shock occurred in the proventriculus and fat body (data not presented). By 21 h after the 38.5 °C heat shock, much of the gut tissue stained with Trypan Blue, as shown by the extreme individual depicted in Fig. 6C. The midgut, hindgut and Malpighian tubules stained significantly more at this time than immediately after heat shock (Fig. 7), while in all types of tissue, differences between the level of staining in control larvae and in those 21 h after heat shock were significant (Mann–Whitney U-tests, P<0.05). Large internal necroses also become evident in some larvae.

Fig. 7.

Quantification of Trypan Blue staining, an indicator of tissue damage, among control larvae (open columns), larvae exposed to 38.5 °C for 1 h (stippled columns) and those exposed to 38.5 °C for 1 h and thereafter maintained at 25 °C for 21 h (filled columns). Scoring for each individual was (0) no color, (1) any blue, (2) darkly stained nuclei, (3) large patches of darkly stained cells, and (4) complete staining of most cells in the tissue. Each datum was a composite score for groups of three larvae that were dissected, stained and visually scored together, with half units representing intermediate degrees of staining after averaging within each group. Scoring of hindgut may have included the most posterior section of the midgut in some samples.

Fig. 7.

Quantification of Trypan Blue staining, an indicator of tissue damage, among control larvae (open columns), larvae exposed to 38.5 °C for 1 h (stippled columns) and those exposed to 38.5 °C for 1 h and thereafter maintained at 25 °C for 21 h (filled columns). Scoring for each individual was (0) no color, (1) any blue, (2) darkly stained nuclei, (3) large patches of darkly stained cells, and (4) complete staining of most cells in the tissue. Each datum was a composite score for groups of three larvae that were dissected, stained and visually scored together, with half units representing intermediate degrees of staining after averaging within each group. Scoring of hindgut may have included the most posterior section of the midgut in some samples.

In whole-body lysates, the pattern of Hsp70 expression following a potentially lethal heat shock (i.e. 1 h at 38.5 °C) contrasts with that occurring after a 1 h treatment at 36 °C, which is seldom lethal. After exposure to the higher temperature, Hsp70 expression in third-instar larvae is initially very low, but increases markedly for many hours afterwards. The milder stress, in contrast, leads to maximal expression in whole-body extracts after 1 h of treatment, approaching 70 % of a standard (Feder et al. 1996; Krebs and Feder, 1997), but concentrations decline rapidly afterwards, and are virtually undetectable 21 h after the 36 °C exposure. Similar expression patterns are known for other life stages (Feder et al. 1996) and for tissue culture cells derived from embryos (Solomon et al. 1991).

Hsp70 expression in whole larvae is the net result of differential expression in various tissues, which differ in their kinetics of Hsp70 expression during stress and recovery from stress. Fig. 2 exemplifies this finding. Brain, salivary gland, imaginal disks and hindgut respond quickly during stress and are responsible for the high levels of Hsp70 that may be found within minutes of a stress encounter. However, fat body, which makes up a large portion of larvae late in development, may account for much of the Hsp70 expressed later after heat shock. Coincidentally, those tissues showing rapid induction of Hsp70 all have a common embryonic origin in the ectoderm (Martinez Arias, 1993). Tissues that express Hsp70 more slowly, however, have disparate embryonic origins that include mesoderm (fat body), endoderm (caeca and midgut) and ectoderm (Malpighian tubules) (Skaer, 1993).

The timing of the appearance of Hsp70 varies both within individual cells and in cell types within tissues. Gut cells show a fluorescent signal in the cytoplasm, but not in the nuclei, 4 h after a 36 °C exposure. The transition from nuclear to cytoplasmic localization of Hsp70 correlates with a resumption of synthesis of normal cellular proteins (Velazquez and Lindquist, 1984; Palter et al. 1986). Malpighian tubules comprise two distinct cell types, of which stellate cells express Hsp70 first. The more numerous broad cells peak in Hsp70 expression well after most other tissues. This delay in Hsp70 expression may explain the observations of Singh and Lakhotia (1995), who reported that the broad cells of the Malpighian tubules do not produce Hsp70.

Extreme heat shock (38.5 °C) produced more extensive inter-tissue variation in Hsp70 expression than did 36 °C heat shock (Figs 3–5), although basic patterns followed those described for Fig. 2. After the more intense heat shock, most tissues initially exhibited little Hsp70 staining, which is consistent with the Hsp70 levels reported for whole-larval lysates. In contrast, brain, salivary glands, imaginal disks and hindgut fluoresced intensely at this time.

Experimental increases and decreases in Hsp70 concentration affect thermotolerance correspondingly (Solomon et al. 1991; Welte et al. 1993; Feder et al. 1996). Our expectation a priori, therefore, was that those tissues expressing the highest levels of Hsp70, or those expressing Hsp70 rapidly, should be most resistant to thermal stress. In general, those tissues of D. melanogaster that produced Hsp70 rapidly in response to high temperatures, particularly brain and salivary glands, suffered less damage from heat shock than those expressing Hsp70 slowly, e.g. caeca, and midgut. Trypan Blue staining, a measure of tissue damage, increased in all tissues after heat stress. In non-gut tissues, staining was spotty, suggesting that outright damage or death occurred in a small proportion of cells. The gut, however, incorporated much more Trypan Blue, suggesting extensive necrosis. Gut function is not essential for acute survival, and gut damage may kill only slowly as it deprives larvae of nutrients and water. Indeed, deaths from a 1 h heat shock at 38.5 °C occur 24–48 h after the stress exposure in first-and third-instar larvae, and few larvae die during the actual stress treatment (Krebs and Feder, 1997). Rarely do larvae resume feeding, despite crawling along the food surface and occasionally pupating.

In mammals, Hsp70 expression may vary within specific tissues, for example, in the central nervous system (CNS) (Manzerra and Brown, 1992), but whether low levels of expression correlate with heat susceptibility is not known. In one test of heat-induced protein denaturation, proteins in a gut tissue, liver, denatured before those of muscle or eye lens (Ritchie et al. 1994); the CNS was not examined. Identifying nerve damage is important, however, because CNS failure could debilitate the function of an organ, with the cause attributed incorrectly to the organ itself. Two different transcription factors, HSF1 and HSF2, which differ in their response to heat and thus in their affects on Hsp induction, contribute to the inter-tissue variation (Marcuccilli et al. 1996; Brown and Rush, 1996). D. melanogaster possess only one HSF, but the fact that larval rearing temperatures may affect Hsp70 expression patterns in some D. melanogaster cell types (Hochstrasser, 1987) and the increased tissue variability at higher temperatures found in this study indicate that multiple factors affect Hsp70 regulation.

Our results make several important contributions. (1) Although all tissues express Hsp70 in response to heat, different tissues vary in the timing of their response. (2) Most Hsp70 expression activated by severe heat shock occurs after a return to physiologically normal temperatures, suggesting either that Hsp70 expression responds to the heat-induced damage rather than to the heat shock itself or that Hsp70 expression is itself damaged by heat. (3) From Trypan Blue staining after heat shock, gut tissues appear to be especially sensitive to heat. (4) Covariation among and perhaps even within tissues for expression of Hsp70 and Trypan Blue staining affirms the link between Hsp70 expression and tissue damage, thereby strengthening the evidence for the use of Hsp70 as a biomarker in environmental monitoring (Ryan and Hightower, 1996). It is unclear from the present investigation, however, whether the expression of Hsp70 after heat shock is more symptomatic of cell damage than of a system for repair. In contrast, Hsp expression before heat shock clearly enhances survival (Krebs and Feder, 1997). These results lay the groundwork for an examination of how differential sensitivity of the tissues results in whole-organism thermotolerance, and target the gut for future attention. As gut tissues appear to be the most sensitive to heat shock and the slowest to produce Hsp70, we predict that increasing Hsp70 levels experimentally (Feder et al. 1996) ought to increase the tolerance of these tissues more than others, and that enhanced thermotolerance of the gut should enhance whole-organism thermotolerance.

We thank Susan Lindquist and Mark Martindale for use of microscopes, Julie Feder and Steve Irvine for critically reading the manuscript and/or assistance with microscopy techniques, and Pratumtip Boontrakulpoontawee Maddux for demonstrating her technique for larval dissection. Research was supported by National Science Foundation grants IBN94-08216 and BIR94-19545, and the Louis Block Fund of the University of Chicago.

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