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°C, all were killed by a direct 1 h exposure to −16°C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4°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°C improved their subsequent tolerance of −14 and −16°C. In response to heat stress, fewer than 20% of the bugs survived a 1‐h exposure to 46°C, and nearly all were killed at 48°C. Dehydration, heat acclimation at 30°C for 2 weeks and rapid heat hardening at 37°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.     

Natural and Genetic Engineering of the Heat-Shock Protein Hsp70 in Drosophila melanogaster: Consequences for Thermotolerance

SYNOPSIS. Larvae of the fruit fly, Drosophila melanogaster,live within necrotic fruit, a challenging environment in whichlarvae can experience severe thermal stress. One response tothermal stress, the expression of heat-shock proteins (Hsps),has evolved distinctively in this species; the gene encodingHsp70 has undergone extensive duplication and accounts for thebulk of Hsps that are expressed upon heat shock. Genetic engineeringof hsp70 copy number is sufficient to affect thermotoleranceat some (but not all) life stages. Increases in Hsp70, moreover,can protect intact larvae against thermal inactivation of theenzyme alcohol dehydrogenase and thermal inhibition of feeding.Deleterious consequences of high levels of Hsp70, however, maylimit further evolutionary proliferation of hsp70 genes. Thesefindings illustrate how the perspectives of integrative andcomparative biology, if applied to even well-studied model organisms,can lead to novel findings.     

Three heat shock proteins are essential for rotifer thermotolerance

Heat shock proteins (HSPs) are important molecules in the stress response of organisms from prokaryotes to mammals, and thus may be useful biomarkers for environmental stress. Here we characterize the functional roles of genes belonging to four distinct families of HSPs (hsp40, hsp60, hsp70, and hsp90) in the monogonont rotifer Brachionus manjavacas. Because B. manjavacas inhabits ponds of varying thermal regimes, including ephemeral ponds that may experience temperature fluctuations, HSP-mediated thermotolerance likely is important to its survival and adaptation. Using interference RNA (RNAi), we provide the first conclusive evidence that HSPs are required for rotifer survival following heat stress. Effective RNAi-mediated suppression of all hsp genes except hsp90 was verified via quantitative PCR. Hsp40, hsp60, and hsp70 are required for rotifer thermotolerance (P < 0.05); however, our data do not indicate hsp90 is essential. Quantitative PCR further revealed immediate up-regulation of hsp40 mRNA following heat stress. Additionally, we demonstrated expression of hsp40 mRNA in multiple tissues using fluorescent in situ hybridization. Our characterization of mRNA expression and functional roles for four distinct hsp genes provides a baseline for molecular-level comparisons of the stress response of rotifers with other taxonomic groups, and the technique for in-depth studies of the role of specific genes in rotifer stress responses. Considering the potential for ambient temperatures to impact species survival, competitive interactions, and body size of individuals, thermotolerance may be an important influence on zooplankton community structure.     

Response of heat shock protein genes of the oriental fruit moth under diapause and thermal stress reveals multiple patterns dependent on the nature of stress exposure

Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.     

Differential expression of heat-shock proteins Hsp70 and Hsp90 in vegetative and reproductive tissues of Iris pumila

Tissue-specific variation in Hsp70 and Hsp90 expression was studied in vegetative (leaf) and reproductive organs (floral tube, ovary and stamen) of Iris pumila plants originating from a sun-exposed and a shaded natural population, which experienced similar growth conditions in an experimental garden. Western blot analysis revealed the presence of both the Hsps in all examined tissues, but at different amounts. In addition to Hsp90a and Hsp90b that were previously detected in vegetative tissues, three new immunospecific bands, designated herein as Hsp90c, Hsp90d and Hsp90e, were recognized with the same anti-Hsp90 antibody in the reproductive tissues. Apart from showing tissue-specific differences in the relative amount of Hsp70 and Hsp90, our study provides evidence that the degree of Hsps expression within the same tissue also depended on the habitat type that the I. pumila plants were derived from.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Dehydration,rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, <Emphasis Type="Italic">Belgica antarctica</Emphasis>

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.     

Three Heat Shock Protein Genes from Bactrocera (Tetradacus) minax Enderlein: Gene Cloning,Characterization, and Association with Diapause

Bactrocera (Tetradacus) minax Enderlein is a major pest to wild and cultivated species of citrus. Bactrocera minax produces one generation per year with a long pupal diapause period of over 6 months, which hinders efforts to obtain vast numbers of insects under standard room conditions. Determining the mechanisms of diapause is significantly important for obtaining large quantities of these insects. To characterize the heat shock protein (Hsp) genes of B. minax and to unravel their potential contribution to diapause, we performed 3′ and 5′ RACE to isolate the complementary DNA (cDNA) sequences, bioinformatics to examine the phylogenetic relationships, and real-time quantitative PCR to detect the expression patterns of three Hsp genes during various developmental stages. These results represent the first characterization of the three Hsp genes of B. minax; the open reading frames of Bmhsp23, Bmhsp70, and Bmhsp90 were 510, 1,911, and 1,089 bp, encoding 170, 636, and 363 amino acids, respectively. BmHsp70 and BmHsp90 displayed high identity to previously identified Hsp70 and Hsp90 genes, respectively. BmHsp23 displayed varying similarity, from 28 to 83%, to previously identified small Hsps. Bmhsp23 messenger RNA (mRNA) expression was found to be upregulated during diapause initiation, maintenance, and termination. Bmhsp70 mRNA expression peaked during diapause initiation. Bmhsp90 mRNA expression remained at a relatively low level during deep diapause. Our present results suggest that Bmhsp70 might play an important role in diapause initiation, while Bmhsp23 in diapause initiation and maintenance and Bmhsp90 in diapause regulation. These results improve our understanding of the mechanism of diapause in B. minax at the molecular level.