Joseph P. Rinehart

Up-regulation of heat shock proteins is essential for cold survival during insect diapause

Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the flys heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupas ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects.

Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis

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 degrees C heat shock, while hsc70 was unaffected by the heat stress. In contrast, both hsp70 and hsc70 were upregulated in nondiapausing flies following a -10 degrees C cold shock. In diapausing pupae, hsp70 was highly upregulated during diapause, even at a non-stress temperature of 20 degrees 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.

Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect.

Antarcticas terrestrial environment is a challenge to which very few animals have adapted. The largest, free-living animal to inhabit the continent year-round is a flightless midge, Belgica antarctica. Larval midges survive the lengthy austral winter encased in ice, and when the ice melts in summer, the larvae complete their 2-yr life cycle, and the wingless adults form mating aggregations while subjected to surprisingly high substrate temperatures. Here we report a dichotomy in survival strategies exploited by this insect at different stages of its life cycle. Larvae constitutively up-regulate their heat shock proteins (small hsp, hsp70, and hsp90) and maintain a high inherent tolerance to temperature stress. High or low temperature exposure does not further up-regulate these genes nor does it further enhance thermotolerance. Such “preemptive” synthesis of hsps is sufficient to prevent irreversible protein aggregation in response to a variety of common environmental stresses. Conversely, adults exhibit no constitutive up-regulation of their hsps and have a lower intrinsic tolerance to high temperatures, but their hsps can be thermally activated, resulting in enhanced thermotolerance. Thus, the midge larvae, but not the adults, have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat that is more thermally stable than that of the adults but a habitat subjected frequently to freeze-thaw episodes and bouts of pH, anoxic, and osmotic stress.

Heat‐shock protein 90 is down‐regulated during pupal diapause in the flesh fly, Sarcophaga crassipalpis, but remains responsive to thermal stress

Heat‐shock protein 23 (hsp23) and hsp70 are both known to be strongly up‐regulated during pupal diapause in the flesh fly Sarcophaga crassipalpis. This prompted us to investigate whether hsp90 was also up‐regulated during diapause. To test this possibility, we developed a partial clone of a hsp90 family member for use as a probe in Northern blot hybridization. Both high and low temperature exposure up‐regulated hsp90 transcripts in nondiapausing individuals. In contrast to hsp23 and hsp70, hsp90 was down‐regulated following entry into diapause, and returned to prediapause levels after diapause termination. The response of hsp90 to heat shock and cold shock remained intact during diapause: both shocks evoked elevated expression. The results indicate differential regulation of hsps during diapause and in response to thermal injury inflicted on diapausing pupae.

Desiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae

SUMMARY Heat shock proteins (Hsps) are a ubiquitous component of the cellular response to stress in both prokaryotic and eukaryotic organisms, but their role and function during desiccation stress in terrestrial arthropods has received limited attention. Molecular responses to rehydration are arguably as important as those to desiccation in maintaining cellular integrity and enzyme activity, but the role of Hsps during stress recovery is poorly understood and has never been addressed with respect to rehydration in insects. This study identifies distinct differences in the Hsp response to desiccation and rehydration in the flesh fly Sarcophaga crassipalpis, as well as differences in the desiccation responses of diapausing and nondiapausing pupae. In nondiapausing pupae, the expression of two inducible Hsps (Hsp23 and Hsp70) is upregulated by desiccation, but the water loss threshold for Hsp expression changes at different rates of dehydration. Continued desiccation results in the prolonged expression of both Hsp23 and Hsp70, which may contribute to the delayed adult eclosion noted in samples desiccated for more than 3 days at <5% relative humidity/25°C. In diapausing pupae, hsp23 and hsp70 transcripts are already highly expressed and are not further upregulated by desiccation stress. Both of the constitutive Hsps investigated, Hsp90 and Hsc70, were unresponsive to desiccation in both nondiapausing and diapausing pupae. However, both Hsp90 and Hsc70 were upregulated upon rehydration in nondiapausing and diapausing pupae. These results indicate distinct roles for the different Hsps during desiccation stress and rehydration/stress recovery. The response to desiccation recovery (rehydration) is similar to the Hsp response to cold recovery identified in S. crassipalpis: Hsp90 and Hsc70 are upregulated in both cases.

Rapid cold-hardening increases the freezing tolerance of the Antarctic midge Belgica antarctica

SUMMARY Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to -10°C for 24 h; by contrast, nearly all cold-acclimated larvae survived -10°C, and a significant number could survive -15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to -5°C for 1 h prior to exposures to -10, -15 or -20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10°C for 1 h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In cold-acclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20°C for 24 h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance.

Dehydration, rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, Belgica antarctica

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene’s responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.

Desiccation elicits heat shock protein transcription in the flesh fly, Sarcophaga crassipalpis, but does not enhance tolerance to high or low temperatures

Although heat shock protein (hsp) production has been noted in response to multiple environmental stresses, no link has been previously established between desiccation and hsps. Following a nonlethal desiccation at 0% relative humidity (R.H.) for up to 48 h, two heat shock protein transcripts, hsp23 and hsp70, were upregulated in pupae of the flesh fly, Sarcophaga crassipalpis. The transcripts were nearly undetectable in control pupae, but within 24 h after being placed at 0% R.H. high transcript expression was observed. This suggests that protection against desiccation may be linked to the general stress response employed by flesh flies against other environmental stressors such as low or high temperature. Adaptive cross-tolerance to cold shock and heat shock following an hsp-inducing desiccation pretreatment was also tested. Although the two hsp transcripts were upregulated in response to desiccation, the upregulation was less dramatic than the upregulation elicited by heat shock, and desiccation failed to generate tolerance to high or low temperatures.

Stress proteins: A role in insect diapause?

Publisher Summary This chapter reviews a paper which summarizes the association of stress protein gene expression with pupal diapause in flesh flies, and discusses the possible implications of the upregulation of some of these proteins during this time. It links these findings with reports from other species showing the expression of stress proteins during periods of developmental arrest. Heat shock proteins are best known as a highly conserved group of stress proteins that are quickly upregulated in response to environmental stress. Stress proteins with similar sequences are present in organisms as diverse as bacteria, yeast, plants, and humans. The proteins are grouped into several families based on molecular mass. Genes encoding certain stress proteins (Hsp23 and 70) are highly upregulated during diapause, while others are either unaffected (Hsc70), or are downregulated (Hsp90). This disynchrony of expression is in marked contrast to the uniform upregulation of the stress protein genes in response to other stresses, such as heat shock or cold shock. The diapause upregulation of the genes may be linked to a cryoprotective function of the proteins or a possible role in shutting down the cell cycle. The involvement of stress proteins in the dormancies of other animals and plants suggests a conserved mechanism contributing to the arrest of development.

Cryoprotective dehydration and the resistance to inoculative freezing in the Antarctic midge, Belgica antarctica

SUMMARY During winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7–8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to– 3°C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by ∼40% and depressed the body fluid melting point more than threefold to –2.6°C. This melting point depression was the result of the concentration of existing solutes (i.e. loss of body water) and the de novo synthesis of osmolytes. By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the insects intimate contact with environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter.