Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation and deacetylation as regulators of this insect's developmental trajectory.

Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding.

We cloned the gene that encodes prothoracicotropic hormone (PTTH) in the northern house mosquito, Culex pipiens, and investigated its expression profile in short-day (diapause-destined) and long-day (nondiapause-destined) individuals from the fourth-instar larval stage to 2 months of adulthood, as well as after a blood meal. The deduced C. pipiens PTTH (Cupip-PTTH) amino acid sequence contains seven cysteines with a specific spacing pattern. Sequence alignment suggests that Cupip-PTTH is 23% identical to Drosophila melanogaster PTTH, but is ≥59% identical to the PTTHs of other mosquitoes. Cupip-PTTH has structural characteristics similar to those of Bombyx mori PTTH and some vertebrate nerve growth factors with cysteine-knot motifs. PTTH transcripts exhibit a daily cycling profile during the final (fourth) larval instar, with peak abundance occurring late in the scotophase. The fourth-larval instar stage is one day longer in short-day larvae than in long-day larvae, resulting in larger larvae and adults. This additional day of larval development is associated with one extra PTTH cycle. No cycling was observed in pupae, but PTTH transcripts were slightly higher in short-day pupae than in long-day pupae throughout much of the pupal stage. PTTH expression persisted at a nearly constant level in diapausing adult females for the first month but then dropped by ∼50%, while expression decreased at the beginning of adulthood in nondiapausing females and then remained at a low level as long as the females were denied a blood meal. However, when nondiapausing females were offered a blood meal, PTTH transcripts rose approximately 7 fold in 2 h and remained elevated for 24 h. A few diapausing females (∼10%) will take a blood meal when placed in close proximity to a host, but much of the blood is ejected and such meals do not result in mature eggs. Yet, elevated PTTH mRNA expression was also observed in diapausing females that were force fed. Our results thus point to several distinctions in PTTH expression between short-day and long-day mosquitoes, but both types of females responded to a blood meal by elevating levels of PTTH mRNA.

RNA interference directed against ribosomal protein S3a suggests a link between this gene and arrested ovarian development during adult diapause in Culex pipiens.

Arrested ovarian development is a key characteristic of adult diapause in the mosquito Culex pipiens. In this study we propose that ribosomal protein S3a (rpS3a), a small ribosomal subunit, contributes to this shutdown. RpS3a is consistently expressed in females of C. pipiens that do not enter diapause, but in females programmed for diapause, expression of the rpS3a transcript is dramatically reduced for a brief period in early diapause (7-10 days after adult eclosion). RNA interference directed against rpS3a in nondiapausing females arrested follicle development, mimicking the diapause state. The effect of the dsRNA injection faded within 10 days, allowing the follicles to grow again, thus the suppression of rpS3a caused by RNAi did not permanently block ovarian development, implying that a brief suppression of rpS3a is not the only factor contributing to the diapause response. The arrest in development that we observed in dsRNA-injected females could be reversed with a topical application of juvenile hormone III, an endocrine trigger known to terminate diapause in this species. Though we speculate that many genes contribute to the diapause syndrome in C. pipiens, our results suggest that a shut down in the expression of rpS3a is one of the important components of this developmental response.

Responses of the bed bug, Cimex lectularius, to temperature extremes and dehydration: levels of tolerance, rapid cold hardening and expression of heat shock proteins.

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately -20 degrees C, all were killed by a direct 1 h exposure to -16 degrees C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4 degrees C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1-h exposure to 0 degrees C improved their subsequent tolerance of -14 and -16 degrees C. In response to heat stress, fewer than 20% of the bugs survived a 1-h exposure to 46 degrees C, and nearly all were killed at 48 degrees C. Dehydration, heat acclimation at 30 degrees C for 2 weeks and rapid heat hardening at 37 degrees C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.

Decrease in expression of beta-tubulin and microtubule abundance in flight muscles during diapause in adults of Culex pipiens.

The cDNA encoding beta-tubulin in the mosquito Culex pipiens has high similarity with the beta-tubulins reported in other insects. In this study, we examine expression of this gene and microtubule abundance in relation to diapause and low temperature. While non-diapausing mosquitoes express beta-tubulin highly in their thoracic muscles, expression is quite low during adult diapause. The abundance of microtubules was also much lower in flight muscles of diapausing adults than in flight muscles from non-diapausing individuals, as confirmed by laser confocal microscopy of tubulins stained using indirect immunofluorescence. Low temperatures decreased microtubule abundance in midguts of non-diapausing mosquitoes, but microtubule abundance in diapausing mosquitoes was already low and remained unchanged by low temperature exposure. Overall, pixel intensity averages were higher in the flight muscles than in the midguts, and again low temperatures decreased microtubule abundance in the flight muscles of non-diapausing females, while levels remained consistently low in diapausing females. These results clearly indicate that a decrease in microtubule abundance is evoked both by the programming of diapause and, in non-diapausing females, by exposure to low temperatures. Quite possibly the reduced microtubule abundance in the flight muscles and reduced expression of beta-tubulin are functionally correlated to the reduction in flight activity that is associated with low temperature and diapause.

A shut-down in expression of an insulin-like peptide, ILP-1, halts ovarian maturation during the overwintering diapause of the mosquito Culex pipiens.

Short day-length is used to programme adult diapause in the mosquito, Culex pipiens. The downstream endocrine event that halts ovarian maturation is a shut-down in juvenile hormone (JH) production, and recent evidence suggests that the insulin signalling pathway may be a key upstream player in executing this developmental arrest. Genes encoding insulin-like peptides-1, -2 and -5 were identified in C. pipiens, and we report that transcript levels of insulin-like peptides-1 and -5 were significantly lower in diapausing females than in their nondiapausing counterparts. Genes encoding both insulin-like peptides-1 and -5 were suppressed using RNA interference in mosquitoes programmed for nondiapause, and ovarian maturation was monitored. Knocking down insulin-like peptide-1 with RNAi in nondiapausing mosquitoes resulted in a cessation of ovarian development akin to diapause, and this arrest in development could be reversed with an application of JH. Knocking down insulin-like peptide-5 did not alter ovarian development. These results are consistent with a role for insulin-like peptide-1 in the signalling pathway leading from the perception of short day-lengths to the shut-down in JH production that characterizes adult diapause in C. pipiens.

Rapid cold hardening elicits changes in brain protein profiles of the flesh fly, Sarcophaga crassipalpis.

Rapid cold hardening (RCH) refers to the enhanced cold tolerance acquired by a brief exposure to a moderately low temperature. Although ecological aspects of this response have been well documented in insects, less is known about the physiological and biochemical mechanisms elicited by RCH. In this study we used two-dimensional electrophoresis to detect differences in brain protein abundance in pharate adults of the flesh fly Sarcophaga crassipalpis, in response to a 2 h RCH exposure at 0 degrees C. Fourteen high abundance proteins that responded to RCH were selected for mass spectrometric identification. Three proteins that increased in abundance during RCH included ATP synthase subunit alpha, a small heat shock protein (smHsp), and tropomyosin-1 isoforms 33/34. Eleven proteins that decreased in abundance or were missing following RCH included several proteins involved in energy metabolism, protein degradation, transcription, actin binding, and cytoskeleton organization. That several proteins increased in abundance during RCH underscores the dynamics of the RCH mechanism and suggests that more than one physiological response likely contribute to RCH. The increase in ATP synthase suggests an elevation of ATP during RCH, and the smHsp increase suggests that at least one of the Hsps is actually mobilized during RCH, rather than after RCH as previously assumed.

Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause.

Most molecular work on insect diapause has focused on the expression of unique diapause transcripts, rather than the protein products. Here we present results from a proteomic comparison of diapausing and nondiapausing pupal brains. Proteins extracted from diapausing pupal brains of the flesh fly Sarcophaga crassipalpis were separated by two-dimensional gel electrophoresis and compared with those from nondiapausing pupal brains. Unique proteins and proteins present at different levels of abundance in diapausing and nondiapausing brains were identified by Nano-LC/MS/MS (capillary-liquid chromatography-nanospray tandem mass spectrometry). With this approach and Coomassie staining, we detected 37 diapause-unique or upregulated (> or = 2x) proteins, and 43 proteins that were downregulated or not present in diapause. Heat shock proteins (Hsp70 and several small Hsps) were among the most conspicuous brain proteins present in higher amounts during diapause. Brain proteins that were less abundant in diapause included phosphoenolpyruvate synthase, fatty acid binding protein, EG0003.7, and an endonuclease. Our 2-D proteome maps included several additional unknown proteins that were more abundant in either the diapause or nondiapause brains. While the mRNAs encoding some of these proteins (e.g. Hsps) were previously known to be associated with diapause, the other proteins were not known to be linked to diapause, thus suggesting that the proteomic approach nicely supplements the work done at the transcript level.

Habitat requirements of the seabird tick, Ixodes uriae (Acari: Ixodidae), from the Antarctic Peninsula in relation to water balance characteristics of eggs, nonfed and engorged stages.

The seabird tick Ixodes uriae is exposed to extreme environmental conditions during the off-host phase of its life cycle on the Antarctic Peninsula. To investigate how this tick resists desiccation, water requirements of each developmental stage were determined. Features of I. uriae water balance include a high percentage body water content, low dehydration tolerance limit, and a high water loss rate, which are characteristics that classify this tick as hydrophilic. Like other ticks, I. uriae relies on water vapor uptake as an unfed larva and enhanced water retention in the adult, while nymphs are intermediate and exploit both strategies. Stages that do not absorb water vapor, eggs, fed larvae and fed nymphs, rely on water conservation. Other noteworthy features include heat sensitivity that promotes water loss in eggs and unfed larvae, an inability to drink free water from droplets, and behavioral regulation of water loss by formation of clusters. We conclude that I. uriae is adapted for life in a moisture-rich environment, and this requirement is met by clustering in moist, hydrating, microhabitats under rocks and debris that contain moisture levels that are higher than the tick's critical equilibrium activity.

Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence.

Distinct differences in the temporal expression patterns of genes associated with pupal diapause were noted in the flesh fly, Sarcophaga crassipalpis. The first change observed was a decline in expression of the gene encoding heat shock protein 90 (hsp90) 2 days after pupariation (1 day before the pupa reaches the phanerocephalic stage characteristic of diapause). In contrast, hsp23 and hsp70 transcripts were undetectable in nondiapause samples and d1-d4 diapause-programmed pupae, but were up-regulated just after the start of diapause, 5 days after pupariation. An increase of glycerol content in diapausing pupae was also noted at the start of diapause. The gene encoding proliferating cell nuclear antigen (pcna) was diapause down-regulated, and this occurred in two phases, with the first decline in expression 7 days after pupariation and a second decline in the level of expression on day 14. For pupae held at 20 degrees C for 20 days and transferred to 10 degrees C, diapause ended after 90-100 days at the lower temperature. However, pupae remained in a state of post-diapause quiescence (d100-d150) and sustained diapause-like hsp and pcna expression patterns until adult development was initiated. Glycerol concentrations and survival declined during the post-diapause phase. This study suggests a distinct sequence in the pattern of gene expression at the onset of diapause, but the genes we have monitored do not contribute to the switch to covert developmental potential at the transition from diapause to post-diapause quiescence.

Molecular characterization of prothoracicotropic hormone and diapause hormone in Heliothis virescens during diapause, and a new role for diapause hormone.

cDNAs encoding prothoracicotropic hormone (PTTH) and diapause hormone (DH) were isolated from the tobacco budworm, Heliothis virescens (Hvi). Hvi-PTTH cDNA reveals key structural features known from other PTTHs, yet there is a > 30% amino acid difference between the PTTH sequences found in this noctuid when compared with the PTTHs known from Bombycoidea. Hvi-DH-PBAN cDNA encodes a precursor protein including a DH-like peptide, pheromone biosynthesis-activating neuropeptide (PBAN) and three additional neuropeptides with an FXPRL sequence at the C terminus. PTTH and DH-PBAN transcripts are most highly expressed in the brain and subesophageal ganglion (SG), respectively. Expression of both the PTTH and the DH-PBAN transcripts is high in larvae and remains high in nondiapausing pupae, but in larvae programmed for pupal diapause, expression declines sharply at the onset of larval wandering behaviour and remains low during pupal diapause. This pattern implies that both of these genes are shut down during diapause. These results are not consistent with a role for DH in promoting the entry into diapause as noted in Bombyx mori. Instead, the higher expression of DH in nondiapausing pupae suggests a possible role for DH in promoting continuous development. The injection of DH into diapausing pupae did indeed successfully terminate diapause, thus suggesting a possible new role for this neuropeptide.

Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar.

During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut. We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily. We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species. Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause. This mRNA pattern reflects the pattern of protein synthesis. These results suggest that hemolin is developmentally up-regulated in the gut during diapause. Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA. Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut. Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause. Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.

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.

Sequence and transcription patterns of 60S ribosomal protein P0, a diapause-regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis.

We have isolated and sequenced a 1308bp clone from a pupal brain cDNA library of the flesh fly, Sarcophaga crassipalpis, showing 97% amino acid (aa) sequence similarity to Ceratitis capitata 60S acidic ribosomal protein P0 (CcP0) and 93% aa sequence similiarity to Drosophila melanogaster P0 (DmP0). DmP0 is a multifunctional protein necessary for efficient protein translation of the 60S ribosome as well as DNA repair via AP3 endonuclease activity. In this study, we observed that S. crassipalpis P0 (ScP0) is cyclically regulated throughout the fly's overwintering pupal diapause. Expression of ScP0 cycles out of phase with the 4day cycles of O(2) consumption: the peak day of O(2) consumption is characterized by low ScP0 expression, while high expression is noted during the trough of the O(2) consumption cycle. The O(2) cycles, which are in turn driven by cycles of juvenile hormone (JH), can be eliminated by application of a JH analog (JHA). Pupae rendered acyclic with a JHA application consume O(2) at a constant high rate and ScP0 is consistently downregulated. Our findings thus suggest that the cyclic nature of ScP0 regulation during pupal diapause is linked to the JH-mediated metabolic cycles characteristic of this species.

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

Parturition hormone in the tsetse Glossina morsitans: activity in reproductive tissues from other species and response of tsetse to identified neuropeptides and other neuroactive compounds.

Parturition hormone (PH) activity is present not only in the uterus of the tsetse Glossina morsitans but also in the oviducts of Bombyx mori and Schistocerca gregaria, as well as the ejaculatory duct of S. gregaria males. Activity thus appears to be present in the reproductive ducts of diverse insect taxa. To determine whether any of the common insect neuropeptides are capable of mimicking the effect of PH, 35 identified neuropeptides and analogs were evaluated for PH activity. Modest PH activity was observed for only high doses of proctolin and a pyrokinin analog, thus suggesting that PH is unlikely to be closely related to any of the identified neuropeptides tested. While proctolin was highly effective in stimulating contractions of the S. gregaria oviduct, the extract from the tsetse uterus elicited only a weak response in this bioassay. PH activity was, however, effectively mimicked with an injection of 8 bromo-cyclic GMP, thus suggesting a potential role for this cyclic nucleotide in mediating the PH response. Pregnant females were responsive to PH, other neuropeptides and cyclic nucleotides only when females were neck-ligated. In intact females, the brain can presumably override the stimulation provided by the active compounds.

Cold hardiness of the fly pupal parasitoid Nasonia vitripennis is enhanced by its host Sarcophaga crassipalpis.

Supercooling points (SCPs) and low temperature survival were determined for diapausing and nondiapausing larvae of the ectoparasitoid Nasonia vitripennis. Neither nondiapausing nor diapausing larvae could survive tissue freezing. The SCP profiles were nearly identical for nondiapause-destined (-27 degrees C) and diapausing larvae (-25 degrees C), but these values were not indicative of the lower limits of tolerance in either type of larvae: larvae were killed by chilling at temperatures well above the SCP. Diapausing larvae could withstand low temperature exposures 3-8 times longer than their nondiapausing counterparts. Low temperature survival was enhanced in diapausing and nondiapausing larvae by their encasement within the puparium of the host flesh fly, SARCOPHAGA CRASSIPALPIS: the LT(50)s determined for nondiapausing and diapausing larvae enclosed by fly puparia were 2-3 times higher than values calculated for larvae removed from the puparia. Additional low temperature protection was gained through acquisition of host cryoprotectants during larval feeding: nondiapausing parasitoid larvae that fed on diapausing flesh fly pupae with high levels of glycerol were able to survive exposure to a subzero temperature 4-9 times longer than wasps reared on nondiapausing fly pupae that contained lower quantities of glycerol. Alanine may also contribute to the cold hardiness of N. vitripennis, as evidenced by the fact that larvae feeding on diapausing fly pupae both contained higher concentrations of alanine and exhibited greater cold hardiness. The results thus demonstrate that several critical features of cold hardiness in the wasp are derived from biochemical and physical attributes of the host.

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.

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.