George D. Yocum

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.

UPREGULATION OF A 23 KDA SMALL HEAT SHOCK PROTEIN TRANSCRIPT DURING PUPAL DIAPAUSE IN THE FLESH FLY, SARCOPHAGA CRASSIPALPIS

A diapause upregulated cDNA clone was isolated from a cDNA library generated from brain mRNA of diapausing Sarcophaga crassipalpis pupae. The clone hybridized to a 1600 bp transcript on a northern blot. The insert is 823 bp in length, has a tentative open reading frame of 615 bp, and codes for a 23 kDa protein. The clone has a high level of identity at the amino acid level with the four small heat shock proteins of Drosophila melanogaster. Northern analysis revealed no detectable expression of the transcript in diapause- or nondiapause-programmed wandering larvae, and only trace expression in nondiapausing pupae. But, the transcript was highly expressed beginning at the onset of diapause and continuing throughout diapause. Expression promptly decreased when diapause was terminated. In nondiapausing individuals the transcript was highly expressed in response to cold shock or heat shock, but temperature stress did not cause greater expression in diapausing pupae. The results imply that expression of this small heat shock protein, a response elicited by temperature stress in nondiapausing individuals, is a normal component of the diapause syndrome. The upregulation of this gene during diapause suggests that it plays an essential role during this overwintering developmental arrest.

Cold shock elicits expression of heat shock proteins in the flesh fly, Sarcophaga crassipalpis

Abstract When exposed to brief cold shocks of −10 or −18°C, pharate adults (red eye stage) of the flesh fly, Sarcophaga crassipalpis are unable to eclose as adults. The injury does not cause immediate death: even an 8 h exposure to −18°C allows pharate adult development to proceed to the point of eclosion. Though development continues following cold shock, we observed a dose-dependent decrease in the rate of oxygen consumption, indicating that damage has occurred. The rate of protein synthesis is slow during the first hour after cold shock but quickly recovers. The previously described heat shock proteins, molecular weights 92 and 72 kDa, are expressed in both the brain and integument during recovery from cold shock and are expressed for longer times with increased severity of the cold shock. The cold shock induced 72 kDa protein is immunologically related to the heat shock 70 protein family of Drosophila melanogaster. In addition to the heat shock proteins, three proteins with molecular weights of 78, 45 and 23 kDa, are induced in the integument, but not in the brain, during recovery from cold shock. We previously demonstrated that at high temperatures different developmental stages synthesize two different members of the heat shock 70 protein family, a 65 and a 72 kDa protein. We detect no such developmental switch in response to cold shock. These results demonstrate that heat shock proteins can be induced by extremes of both high and low temperatures but the nature of the stress (heat or cold shock) determines which proteins are induced.

Alteration of the eclosion rhythm and eclosion behavior in the flesh fly, Sarcophaga crassipalpis, by low and high temperature stress

Abstract Heat shock (45°C), cold shock (−10°C), and indirect chilling injury (a prolonged exposure to 2°C) did not interfere with the continuation of pharate adult development in the flesh fly, Sarcophaga crassipalpis, but such flies failed to eclose properly when the exposure was of sufficient duration. In all three forms of injury, development following the temperature treatments was also retarded. Among flies that were less severely affected and still capable of emerging as adults, the circadian time of adult eclosion shifted from near dawn to near the middle of the photophase, thus suggesting that the neurally-based clock is among the systems most vulnerable to heat-shock and cold-shock injury. Tensiometric records of ptilinum expansion revealed important differences in the nature of the injury caused by the different temperature stresses. Heat-shocked flies and those subjected to indirect chilling injury displayed the two behavioral programs normally associated with adult eclosion, the program for obstacle removal (POR) and the program for forward movement (PFM), but such flies failed to eclose because the muscle contractions generated by these motor patterns were insufficient for successful eclosion. In contrast, cold-shocked flies retained the capacity for strong muscle contraction, but the centrally-generated POR and PFM programs were altered. As the duration of cold shock increased, both patterns became more erratic; the PFM program was then lost completely, and in the most severe cases of cold-shock injury, flies lost the capacity to generate both programs. This suggests that neuronal damage is the likely cause of injury inflicted by cold shock.

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.

Diapause specific proteins expressed by the brain during the pupal diapause of the flesh fly, Sarcophaga crassipalpis

Abstract Proteins synthesized by pupal brains of Sarcophaga crassipalpis were examined during diapause and nondiapause conditions using pulse labelling and 2-dimensional electrophoresis. In vitro cultured brains from diapausing pupae synthesized fewer proteins than brains from nondiapausing pupae or pharate adults. Brains from older diapausing pupae (60 days) synthesized slightly more proteins than brains from pupae in early diapause (14 days). A cluster of about 15 brain proteins appears to be specific to diapausing pupae. In contrast, no differences could be detected in the brain proteins of the photosensitive first-instar larvae exposed to long (nondiapause) or short (diapause) daylengths.

Prolonged thermotolerance in the flesh fly, Sarcophaga crassipalpis, does not require continuous expression or persistence of the 72 kDa heat-shock protein

Abstract A brief exposure to a supraoptimal temperature (35–45°C) induced thermotolerance in pharate adults of the flesh fly Sarcophaga crassipalpis . While a 90 min exposure to 45°C was normally lethal to flies reared at 25°C, a brief pretreatment at high temperature generated protection from heat-shock injury. The induction of thermotolerance was dependent upon both the temperature and duration of the pretreatment, and in this study maximal thermotolerance was induced by a 2 h exposure to 40°C. Thermotolerance induced by a 2 h exposure to 40°C decayed slowly over a 72 h period. This prolonged thermotolerance appears to be independent of the continuous synthesis and persistence of heat-shock proteins.

A Fluctuating Thermal Regime Improves Survival of Cold-Mediated Delayed Emergence in Developing Megachile rotundata (Hymenoptera: Megachilidae)

ABSTRACT A significant concern in the commercial application of the alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), for pollination is synchronizing bee emergence and activity with peak crop bloom. Previous studies have demonstrated that the commercial spring incubation of this species can be successfully interrupted by low temperature incubation, thereby slowing development and giving producers flexibility in timing emergence to weather conditions or crop bloom. In this study, we demonstrate that the implementation of a fluctuating thermal regime, during which bees are given a daily one hour pulse of high temperature, markedly increases the “shelf-life” of individuals of this species. Although constant temperatures can be used to store bees for up to 1 wk with no decrease in survival, properly staged bees can be stored for up to 6 wk without a significant decrease in percentage of emergence. Hence, we expect fluctuating thermal regime protocols to become a valuable tool for M. rotundata managers, especially when timing nesting activity with peak bloom to maximize effectiveness.

Interrupted Incubation and Short-Term Storage of the Alfalfa Pollinator Megachile rotundata (Hymenoptera: Megachilidae): A Potential Tool for Synchronizing Bees with Bloom

ABSTRACT A useful technique for synchronizing pollinators with the alfalfa, Medicago savita L. (Fabaceae), bloom is to interrupt the late spring incubation of developing bee pupae and pharate adults of Megachile rotundata (F.) (Hymenoptera: Megachilidae) with short-term low-temperature storage. However, low-temperature exposure can be stressful depending on the temperature, duration of exposure, and the developmental stage exposed. To evaluate the effect of low-temperature storage after development had already been initiated by exposure to 29°C, three developmental stages (eye pigment pupae, body pigment pupae, and pharate adults ready to emerge) were exposed to 6, 12, or 18°C for durations up to 28 d. The effect of delaying the termination of overwintering storage (6°C) from April to July (“April bees” and “July bees,” respectively) also was examined. The following observations were made: 1) All developmental stages of the April bees and July bees examined can be stored without harmful effects, as measured by percentage of survival, for 14 d at 12°C and above. 2) Postponing the termination of overwintering storage at 6°C from April until July significantly decreased the mean number of days to 95% adult emergence in the July ready-to-emerge bees across the three temperatures tested. 3) The increase of overwintering duration also caused a decrease in the postemergence longevity of the ready-to-emerge adults stored at 6°C for 14 d. 4) Of the three storage temperatures examined, 18°C seems to be the optimal storage temperature for short-term storage of developing bees because of their slow but continuing development without increased mortality either during storage or after emergence.