25

32 33 34 35 36 Pretreatment temperature (60 min)

Figure 5.9 (a) The effect of hardening temperature on Hsp70 accumulation and (b) larval thermotolerance (measured as survival of exposure to a high temperature) in excision and extra-copy strains of Drosophila melanogaster.

Source: Reprinted from Journal of Insect Physiology, 44, Krebs and Feder, 1091-1101, © 1998, with permission from Elsevier.

32 33 34 35 36 Pretreatment temperature (60 min)

Figure 5.9 (a) The effect of hardening temperature on Hsp70 accumulation and (b) larval thermotolerance (measured as survival of exposure to a high temperature) in excision and extra-copy strains of Drosophila melanogaster.

Source: Reprinted from Journal of Insect Physiology, 44, Krebs and Feder, 1091-1101, © 1998, with permission from Elsevier.

the excision and extra-copy strains Feder and Krebs (1998) also demonstrated that Hsp70 expression promotes the recovery of alcohol dehydrogenase activity following heat shock.

Excision Extra-copy

Number of pretreatments

Figure 5.10 Larva to adult survival in excision and extra-copy strains of Drosophila melanogaster exposed to repreated hardening treatments, compared to a control strain held at 25°C.

Number of pretreatments

Figure 5.10 Larva to adult survival in excision and extra-copy strains of Drosophila melanogaster exposed to repreated hardening treatments, compared to a control strain held at 25°C.

Source: Feder and Krebs (1998).

The expression of Hsp70, and presumably its protective role, varies genetically and is conserved across life-cycle stages (Krebs et al. 1998). However, the relationship between Hsp70 and thermotolerance is not always readily discernible (Dahlgaard et al. 1998; Goto and Kimura 1998), and in some instances tolerance and Hsp70 expression appear to be relatively independent (Goto et al. 1998; Lansing et al. 2000).

Expression of Hsp70 is not only associated with benefits to the organism. Continuous elevation of Hsp70 levels results in a decline in growth and division of cells, a reduction in larva to adult survival in extra-copy D. melanogaster relative to the excision strain when larvae are repeatedly exposed to hardening temperatures (Fig. 5.10) (Feder and Krebs 1998; Feder 1999), and a decline in egg hatch (Silbermann and Tatar 2000). Likewise, in isofemale lines of wild-caught D. melanogaster there is a negative relationship between Hsp70 expression and survival at constant 25° C (Krebs and Feder 1997). The constitutive presence of Hsp70 can be harmful because it interferes with cell signalling pathways, impedes normal processing and degradation of unfolded proteins, or directs cellular machinery away from synthesizing other proteins to Hsp synthesis (Zatsepina et al. 2001). Although constitutive expression of Hsp70 might also represent a metabolic cost to the organism, so far this idea has not been supported (Krebs and Feder 1998b).

The trade-off between the costs and benefits of Hsp70 expression is the most likely explanation for a decline in the expression of Hsp70 and inducible thermotolerance when either laboratory strains (Sorensen et al. 1999; Lerman and Feder 2001) or wild populations (Sorensen et al. 2001; Zatsepina etal. 2001) evolve at high temperatures. These strains experience most of the deleterious consequences, but derive few benefits from Hsp70 expression because they rarely encounter potentially lethal high temperatures (Zatsepina et al. 2001). This results in downregulation of Hsp70 expression (Sorensen et al. 1999; Lansing et al. 2000), and consequently a decline in inducible thermotolerance, although basal thermotolerance generally increases. While the precise mechanism by which Hsp70 expression is reduced remains incompletely known, Zatsepina et al. (2001) have suggested that the insertion of two transposable elements, H.M.S. Beagle in the 87A7 hsp70 gene cluster, and Jockey in the hsp70Ba gene promoter, are responsible for this reduced expression. In addition, there is some evidence that juvenile hormone plays an important role in the development of the stress response. Exposure of D. melanogaster to stress results in a decline in JH-hydrolysing activity (Gruntenko et al. 2000). Given that JH is known to control reproduction, the continued presence of JH may well explain alterations in fecundity seen in several species in response to stress. Rinehart and Denlinger (2000) also suggested that ecdysteroids may play an important role in the regulation of Hsp90, because ecdysteroids are absent throughout diapause, but are synthesized and released following diapause termination. The time course of these events and those of Hsp90 upregulation are therefore similar. Clearly, the regulation of the stress response by the hormonal system in insects deserves further attention (Denlinger et al. 2001).

Heat shock proteins are not only expressed in response to high temperature stress, but are characteristic of the response of insects to virtually all stresses (Burton et al. 1988; Goto et al. 1998; Feder and Hofmann 1999; Tammariello et al. 1999). For example, larval crowding of Drosophila also induces Hsp70 expression, and leads to correlated responses such as increases in the ability to survive heat shock and increases in longevity (Tatar 1999; Gruntenko et al. 2000; Sorensen and Loeschcke 2001). However, not all stresses that result in expression of heat shock proteins result in a generalized stress response. Tammariello et al. (1999) found that although desiccation in S. crassipalpis resulted in upregulation of hsp23 and hsp70, this response was less dramatic than the upregulation found in response to heat shock. In consequence, desiccation failed to result in tolerance to high or low temperatures. Likewise, upregulation of hsp70 has little effect on knockdown temperature (Sorensen et al. 2001; Sorensen and Loeschcke 2001), but this may be due to the independence of heat shock survival and knockdown survival (Section 5.2.2). In some species, Hsp70 may also be constitutively expressed and does not increase in response to heat stress, or if it does is sufficiently tissue-specific that overall levels do not change (Salvucci et al. 2000). Irrespective of this variation, the most significant questions that remain in Hsp research are whether the responses that have largely been induced in the laboratory are relevant to the field, and whether variation in Hsp expression is related to the variation in thermotolerance found between populations and between species.

Field temperatures, heat shock, and Hsps The importance of determining the relevance of laboratory studies to the field situation was recognized by Feder and his colleagues early on in their investigation of heat shock in Drosophila species. In a series of studies they demonstrated that necrotic fruit in the sun could rapidly reach temperatures that were not only high enough to induce the heat shock response (Fig. 5.11) (Feder 1997), but were also either lethal to larvae and pupae (Feder et al. 1997a) (Fig. 5.4) or induced developmental defects in the emerging adults (Roberts and Feder 1999). These effects resulted largely from an inability of ovipositing females to respond to cues that might indicate past (and thus future) high fruit

1000 1100 1200 1300 1400

Time of day

Figure 5.11 Temperatures of necrotic peaches exposed to full sunlight and to shade, and air temperature measured at the same time. Source: Feder et al. (1997). Functional Ecology 11, 90-100, Blackwell Publishing.

1000 1100 1200 1300 1400

Time of day

Figure 5.11 Temperatures of necrotic peaches exposed to full sunlight and to shade, and air temperature measured at the same time. Source: Feder et al. (1997). Functional Ecology 11, 90-100, Blackwell Publishing.

temperatures (Feder et al. 1997b). Feder and colleagues also showed that under field conditions (or simulated field conditions) enhanced expression of Hsp70 associated with the transgenic extra-copy strains could increase larval survival (Roberts and Feder 2000) and reduce developmental abnormalities in emerging adults (Roberts and Feder 1999). Although it seems likely that behavioural avoidance reduces the exposure of adults to high temperature, some adults also show enhanced levels of Hsp70 expression in the field, indicating exposure to potentially lethal temperatures (Feder et al. 2000b).

In a remarkable investigation of Chrysomela aeneicollis (Coleoptera, Chrysomelidae), Dahlhoff and Rank (2000) showed that at low temperatures (20°C) the beetles do not express Hsp70, but that expression increases rapidly with temperatures above 24°C. Temperatures close to 35°C are routinely experienced by beetles in the field, and consequently Hsp70 expression in field-collected beetles is also high. However, this expression varies with both latitude and altitude such that field collected beetles from colder environments express lower levels of Hsp70. Moreover, the northern, low temperature population, which expresses considerably greater levels of Hsp70 under heat stress in the laboratory, has a phosphoglucose isomerase

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