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.     

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.     

Role of cytoplasmic proteins in cold shock injury of Amoeba

Strains of Amoeba have been used to study the mechanisms of cellular injury induced by rapid cooling (cold shock). Cell viability was found to depend on the time and temperature of cold exposure, on the rate of cooling and on the morphology of the cells prior to chilling. All strains underwent a granuloplasmic contraction following undercooling to ?10 °C, although its extent varied; strains most damaged by cold shock exhibited the most violent cytoplasmic contractions. Cryomicroscopy demonstrated that the cellular contraction occurred upon rewarming, not during cooling. Cells damaged by cold shock were osmotically responsive, demonstrating that irreversible damage to the plasmalemma does not account for the phenomenon.Several compounds protected Amoeba against cold shock injury, glycerol and glucose being the most effective. With glycerol an optimum rate of cooling was observed upon cooling to ?10 °C, at both faster and slower cooling rates damage increased.The state of cellular actin in control cells and following cold shock was monitored by the DNase 1 inhibition assay and by electron microscopy. A comparatively “cold shock resistant” strain of A. proteus was found to contain less total actin per unit cellular protein than the more “sensitive” Amoeba sp. strain Bor. In the Bor strain a cold-induced aggregation of cytoplasmic filaments was evident in electron micrographs, presumably a crosslinking of preexisting F-actin.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.     

Cold shock induction of thermal sensitivity in Listeria monocytogenes

Cold shock at 0 to 15 degrees C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60 degrees C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8 degrees C for controls and 7.7 degrees C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28 degrees C followed by heating at 60 degrees C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D(60) values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures

Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.     

Short term increases in the cold tolerance of red osier dogwood stems induced by application of cysteine

Bark tissues of Cornus stolonifera stems, treated with cysteine at 24 hours after treatment, survived exposure to −11 C (the tissue temperature) with little or no injury. An initiation of increase in the cold tolerance was usually observed when plants were treated with cysteine at 12 hours after treatment. Neither plants at 36 or 48 hours after treatment nor plants 12 hours before treatment had shown increases in the cold tolerance. They were killed below −5 C, which was the survival temperature of untreated control plants. Two weeks or more of short day induction before cysteine application were required for a significant effect of short term 5 C increase in the cold tolerance.     

Cold shock injury in Tetrahymena pyriformis

The ciliated protozoan Tetrahymena pyriformis has been used to study the biochemistry of cellular injury induced by rapid cooling (cold shock). Cellular viability was found to depend on the time and temperature of cold exposure, and the rate of cooling. During cooling to −7.5 °C, in the absence of ice, an optimal rate of cooling of 2.5 °C min⁻¹ was observed; at both faster and slower cooling the recovery decreased. Following acclimation at a reduced temprature (10 °C) the viability following rapid cooling was significantly different from that of cultures maintained at 20 °C. Analysis of the phospholipid fatty acids from cells grown at 10 °C demonstrated that, at the reduced temperature, there was an increase in the average degree of fatty acyl unsaturation. Cold-shock injury in Tetrahymena is associated with membrane thermotropic events which are determined by temperature per se, whereas viability is a function of the rate of cooling. A hypothesis of injury is presented in which the presence of gel-phase lipid within the membrane is not the critical event, but it is the pattern of nucleation within the membrane which ultimately determines the extent of cellular injury.     

Increase in cold-shock tolerance by selection of cold resistant lines in Drosophila melanogaster

Abstract.

• 1 In Drosophila melanogaster, the cold-shock tolerance of adult flies at -7°C increased 22% after a prior 2h exposure to 4°C as measured by LD₅₀, the dose (degree minutes of exposure to subzero temperature) which resulted in 50% mortality.
• 2 Cold-shock tolerance was further significantly increased by selecting cold resistant lines by exposure of adults (1) to 4°C for 2 h (short-term chilling), or (2) to -7°C for 80–120 min (cold shock), or (3) to short-term chilling followed by cold-shock.
• 3 After ten generations of selection, the greatest increase in cold-shock tolerance was found in flies selected using the combined exposure of short-term chilling and cold shock. LD₅₀s increased 33% in comparison with the unselected control strain when no chilling pre-treatment was given prior to cold shock at -7°C.
• 4 The rapid cold-hardening response increased 82% in the line selected by the short-term chilling and cold-shock regime.
• 5 The enhanced cold-shock tolerance was relatively stable since no decrease was observed after four generations without selection.
• 6 This report shows the role of short-term adaptation as well as selection in the capacity to survive low temperatures in non-diapausing stages of insects.

     

Development of Spalangia cameroni and Muscidifurax raptor (Hymenoptera: Pteromalidae) on live house fly (Diptera: Muscidae) pupae and pupae killed by heat shock, irradiation, and cold

The objective of this study was to evaluate the suitability of killed house fly (Musca domestica L) pupae for production of two economically important pupal parasitoids. Two-day-old fly pupae were subjected to heat shock treatments of varying temperatures and durations in an oven at >or=70% RH; exposure to temperatures of 55 degrees C or higher for 15 min or longer resulted in 100% mortality. Exposure to 50 degrees C resulted in 40 and 91% mortality at 15 and 60 min, respectively. All (100%) pupae placed in a -80 degrees C freezer were killed after 10-min exposure; exposure times of <5 min resulted in <21% mortality. Progeny production of Spalangia cameroni Perkins and Muscidifurax raptor Girault and Sanders (Hymeoptera: Pteromalidae) from pupae killed by heat shock or 50 kR of gamma radiation was not significantly different from production on live hosts on the day when pupae were killed. Freeze-killed pupae produced 16% fewer S. cameroni than live pupae and an equivalent amount of M. raptor progeny on the day when pupae were killed. When killed pupae were stored in freezer bags at 4 degrees C for 4 mo, heat-killed, irradiated, and freeze-killed pupae remained as effective for production of M. raptor as live pupae. Production of S. cameroni on heat-killed and irradiated pupae was equal to parasitoid production on live pupae for up to 2 mo of storage, after which production on killed pupae declined to 63% of that observed with live pupae. Production of S. cameroni on freeze-killed pupae was 73-78% of production using live pupae during weeks 2-8 of storage and declined to 41 and 28% after 3 and 4 mo, respectively. Killing pupae by heat shock provides a simple and low-cost method for stockpiling high-quality hosts for mass-rearing both of these filth fly biological control agents.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.     

Cold shock during liquid production increases storage shelf-life of Cryptococcus nodaensis OH 182.9 after air-drying

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60-70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C.     

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.     

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999     

Modelling the time–temperature relationship in cold injury and effect of high-temperature interruptions on survival in a chill-sensitive collembolan

1. Temperature- and time-dependent mortalities were studied and modelled in insects exposed in regimes with constant and alternating temperatures. In these experiments, freezing was not a cause of death.
2. Survival rates at a range of constant low temperatures (– 5 to + 1 °C) and for different exposure periods (1–14 days) were measured in the summer acclimated springtail Orchesella cincta .
3. Daily interruptions of the cold exposure with short intervals at high temperature reduced mortality or slowed the increase of mortality. This effect was stronger at higher temperature (19 vs 5 and 12 °C) and increased with the duration of the interruption (0·25–2 h).
4. The injury was reversible when the cold exposure was limited to 2 days.
5. Survival in desiccated animals (14% water loss) was reduced.
6. It is suggested that the mortality of summer acclimated springtails is caused by a complex metabolic disorder and membrane changes at low temperatures.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

The cellular apoptosis of murine BW5147 thymoma during cold shock]

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.     

A comparison of the responses of tropical and temperate flies (Diptera: Sarcophagidae) to cold and heat stress

Summary Two flesh fly species from the tropical lowlands (Peckia abnormis and Sarcodexia sternodontis) were more susceptible to both cold-shock and heatshock injury than temperate flies (Sarcophaga crassipalpis and S. bullata) and a fly from a tropical high altitude (Blaesoxipha plinthopyga). A brief (2-h) exposure to 0°C elicits a protective response against subsequent cold injury at–10°C in the temperate flies and in B. plinthopyga but no such response was found in the flies from the tropical lowlands. However, both tropical and temperate flies could be protected against heat injury (45°C) by first exposing them to a mild heat shock (2 h at 40°C). The supercooling point is not a good indicator of cold tolerance: supercooling points of pupae were similar in all species, ranging from–18.9 to–23.0°C, and no differences were found between the tropical and temperate species. Among the temperate species, glycerol, the major cryoprotectant, can be elevated by short-term exposure to 0°C, but glycerol could not be detected in the tropical flies. Low-temperature (0°C) exposure also increased hemolymph osmolality of the temperate species, but no such increase was observed in the tropical lowland species. Adaptations to temperature stress thus differ in tropical and temperate flesh flies: while flies from both geographic areas share a mechanism for rapidly increasing heat tolerance, only the temperate flies appear capable of responding rapidly to cold stress. The presence of a heat shock response in species that lack the ability to rapidly respond to cold stress indicates that the biochemical and physiological bases for these two responses are likely to differ.