The effect of thermal stress on protein composition in dogwhelks (Nucella lapillus) under normoxic and hyperoxic conditions

In this laboratory study, dogwhelks (Nucella lapillus) were collected from the intertidal zone and exposed to 16 °C (ambient), 26.5 °C and 30 °C under normal and hyperoxic conditions respectively. It was shown that there was no thermally induced mortality at 26.5 °C, but that the mortality rate was 40–50% in 30 °C. This mortality rate was reduced to 10% if extra oxygen was provided, indicating that oxygen supply was setting the limit for whole organism thermal tolerance. Tissue samples were then analysed for protein features using two-dimensional gel electrophoresis, and both up and down regulation of proteins were visualised by silver staining and crosswise comparisons of gels from control vs. treated animals. The results clearly show that the protein profiles from dogwhelks exposed to increased water temperatures differ from those of the control, but that increased oxygen availability alleviates these differences thus increasing the similarity between heat-shocked and control animal protein pattern. This implies a more stable protein metabolism and might explain the increased survival of heat-shocked individuals when extra oxygen is supplied.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich ascites tumour cells

The rate of polypeptide synthesis is inhibited by 80% in Ehrlich cells incubated at 43 degrees C compared to those at 37 degrees C. The regulatory site of translation resides at polypeptide chain initiation. Polypeptide synthesis does not recover at the higher temperature; however, the inhibition is reversed by returning the cells to 37 degrees C. Neither new RNA synthesis or protein synthesis is required for recovery at 37 degrees C, eliminating degradation of mRNA and irreversible denaturation of a protein essential for polypeptide chain initiation. The concentration of 40-S initiation complexes was found to be reduced markedly in heat-shocked cells compared to controls. This was confirmed in the cell-free protein-synthesizing systems prepared from heat-shocked and control cells. Reversible alteration in the activity of components affecting eIF2 function is, therefore, a likely mechanism of regulation in heat-shocked Ehrlich cells. In extracts from heat-shocked cells, Met-tRNA synthetase activity was unaltered compared to control extracts.     

Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells

Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.     

Effects of temperature,salinity and aerial exposure on predation and lysosomal stability of the dogwhelk Thais (Nucella) lapillus (L.)

The ingestion and absorption rate of standard length Thais lapillus (L.) stepwise-acclimated to constant temperature-salinity conditions and preying on Mytilus edulis (L.) varied directly with environmental salinity at 10, 15 and 20°C. Dogwhelk ingestion and absorption rates indicate that cold torpor existed at 5°C and heat stress was evident at 20°C. The feeding cycle duration was significantly longer for dogwhelks acclimated to 20%. S than in those acclimated to 30%. S at 10°C even though no significant difference existed between the two groups of snails in the drilling and ingestion or postfeeding phases of the cycle. Ingestive conditioning of dogwhelks to mussels occurred; the duration of the drilling and ingestion and total feeding cycle declined as a function of the number of mussels consumed by a snail. Dogwhelks of all sizes prey on a wide length range of mussels and there is also a high degree of variability in the ingestion rate of snails as a function of their size. A prominent feature of the lack of a relationship between dogwhelk ingestion rate and snail size was that the percentage of nonfeeding snails increased at low salinity and temperature extremes. Digestive-tubule cell lysosomal stability was tested as an index of digestive capability and animal condition; in stepwise-acclimated dogwhelks, it correlated well with their ingestion and absorption rates. The ingestion rate of dogwhelks acclimated to 30%. S and subjected to a 30?17.5?30%. S semidiurnal pattern of fluctuating salinity for 21 days was significantly lower than for snails maintained at 30%. S; however, snails acclimated to 17.5%. S and exposed to the same pattern of fluctuating salinity fed at a higher rate than snails maintained at 17.5%. S. Aerial exposure of snails maintained at 30%. S and 10°C water temperature resulted in an ingestion rate 2.1 times faster than for snails constantly submerged suggesting that tidal emersion is not always stressful to intertidal carnivores. The postfeeding phase of the feeding cycle was shortened in dogwhelks subjected to aerial exposure. Although significant variation occurred in digestive-tubule cell lysosomal stability during the first cycle of fluctuating salinity, the variability had declined significantly by Day 21. This observation suggests that digestive tubule lysosomal stability becomes adapted to a fluctuating osmotic environment, although the initial changes in lysosomal stability are probably related to intralysosomal protein catabolism and production of amino acids for intracellular osmoregulation. Variations in the osmotic environment of T. lapillus have resulted in unexpected outcomes with respect to their ingestion rate under conditions of fluctuating salinity and aerial exposure.     

Ultralow oxygen treatment for control of Latrodectus hesperus (Araneae: Theridiidae) on harvested table grapes

The spider Latrodectus hesperus Chamberlin & Ivie (Araneae: Theridiidae) was subjected to low and ultralow oxygen (ULO) treatments at different temperatures. Complete control of the spiders was achieved in 24-h ULO treatments with 0.5% O2 or lower at 1 degrees C and in a 24-h low oxygen (2%) treatment at 15 degrees C. Oxygen level and temperature greatly affected spider mortality. At 1 degrees C, as oxygen level was decreased from 2 to 0.5%, spider mortality increased from 0 to 100%. At 2% O2, as temperature was increased from 1 to 15 degrees C, spider mortality increased from 0 to 100%. Grape clusters from two table grape (Vitis spp.) cultivars, 'Thompson Seedless' and 'Flame Seedless', were subjected to the 24-h ULO treatment with 0.5% O2 at 1 degrees C. The ULO treatment had no negative effects on grape quality. Because of the relatively short treatment time, effectiveness at low storage temperature and the easily attained oxygen level, we conclude that the ULO treatment have good potential to be implemented commercially for control of black widow spiders on harvested table grapes.     

Hydroprene prolongs developmental time and increases mortality in wandering-phase Indianmeal moth (Lepidoptera: Pyralidae) larvae

Wandering phase Indianmeal moth, Plodia interpunctella (Hübner), larvae were exposed to the label rate of hydroprene (1.9 x 10(-3) mg [AI] /cm2) sprayed on concreted petri dishes. Larvae were exposed for 1, 3, 6, 12, 18, 24, and 30 h and maintained at 16, 20, 24, 28, and 32 degrees C and 57% RH until adult emergence. Larval developmental time and mortality were significantly influenced by temperature and exposure intervals. Maximum developmental time (47.2 +/- 1.3 d) occurred at 16 degrees C, and the minimum developmental time (7.0 +/- 0.5 d) occurred at 32 degrees C. Larval mortality generally increased at all of the five tested temperatures as exposure period increased. The greatest mortality (82.0 +/- 0.1%) occurred when larvae were exposed for 30 h at 28 degrees C, and minimum mortality (0.0 +/- 0.5%) occurred at 16 degrees C when larvae were exposed for 1 h. The relationships between temperature, exposure period, and developmental time were described by polynomial models, based on lack-of-fit tests. Hydroprene has potential to be an effective alternative to conventional insecticides in surface treatments for Indianmeal moth management. Response-surface models derived from this study can be used in simulation models to estimate the potential consequences of hydroprene on Indianmeal moth population dynamics.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.     

Cyclic gas exchange in the giant burrowing cockroach, Macropanesthia rhinoceros: effect of oxygen tension and temperature

The giant burrowing cockroach, Macropanesthia rhinoceros, is endemic to north-eastern Australia and excavates a permanent burrow up to 1m deep into soil. Using flow-through respirometry, we investigated gas exchange and water loss at three different oxygen tensions (21%, 10% and 2% at 20 degrees C) and temperatures (10, 20 and 30 degrees C at 21% oxygen). M. rhinoceros employ cyclic gas exchange (CGE) making the species by far the largest insect known to engage in discontinuous ventilation. CGE featured rhythmic bursts of CO(2) dispersed among inter-burst periods of reduced output. CGE was most commonly observed at 20 degrees C and degraded at <10% oxygen. Mild hypoxia (10% oxygen) resulted in a lengthening of the burst period by approximately two-fold; this result is complementary to oxygen consumption data that suggests that the burst period is important in oxygen uptake. When exposed to severe hypoxia (2% oxygen), CGE was degraded to a more erratic continuous pattern. Also, during severe hypoxia, total water loss increased significantly, although CO(2) release was maintained at the same level as in 21% oxygen. During CGE, an increase in temperature from 10 to 20 degrees C caused both water loss and CO(2) output to double; from 20 to 30 degrees C, CO(2) output again doubled but water loss increased by only 31%.     

Effect of temperature and humidity on insecticidal effect of SilicoSec against Ephestia kuehniella (Lepidoptera: Pyralidae) larvae

Laboratory experiments were carried out to assess the insecticidal effect of the diatomaceous earth formulation SilicoSec against larvae of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), in stored wheat (Triticum durum Desf.). Larvae were exposed to wheat treated with SilicoSec at 400 and 800 ppm and held at 20, 25, and 30 degrees C and 55 and 75% RH. Larval mortality was assessed after 24 h, 48 h, 7 d, and 14 d of exposure in the treated wheat. At both dose rates, mortality increased with temperature, but this increase varied with the exposure interval. At short (< or = 48-h) exposures, larval mortality was significantly higher at 30 degrees C than at the other two temperatures. In contrast, no significant differences were noted between 20 and 25 degrees C. At longer exposures (> or = 7 d), the increase of temperature increased mortality at 800 ppm, but no significant differences were noted between 25 and 30 degrees C at 400 ppm. Furthermore, significant differences in larval mortality were noted between the two humidity levels, but only at exposures > or = 7 d. After 14 h of exposure, at both dose rates examined, the increase of temperature significantly decreased mortality. The results of the current study indicate that E. kuehniella is susceptible to SilicoSec, but temperature and relative humidity should be taken into consideration.     

The interaction of heat and radiation affecting the ability of nuclear DNA to undergo supercoiling changes

DNA damage (putatively strand breaks) from ionizing radiation inhibits the ability of intercalating dyes to induce right-handed supercoils in the DNA loops of HeLa nucleoids [Cook and Brazelle, J. Cell Sci. 22, 287-302 (1976); Roti Roti and Wright, Cytometry 8, 461-467 (1987)] while heat-induced changes in the nuclear matrix enhance this ability [Roti Roti and Painter, Radiat. Res. 89, 166-175 (1982)]. Since heat and radiation interact synergistically or additively on most cellular functions which they affect, the rewinding of DNA supercoils is unusual in that these agents alone affect it in an antagonistic manner. When HeLa cells were exposed to 45 degrees C for 30 min and immediately irradiated with 10 Gy of 137Cs gamma rays, the rewinding response was intermediate between that for cells which had been exposed to 10 Gy only and control. When repair of this damage was assayed in control cells, 97% of the initial damage had been repaired at 30 min postirradiation; at the same time only 10% of the initial damage had been repaired in the heat-shocked cells. This apparent dose reduction effect and the inhibition of repair were interpreted to indicate that heat-induced changes in nuclear structure were masking DNA damage from the assay and the repair system. These effects correlated with the amount of heat-induced excess protein associated with the nucleus and the nucleoid.     

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.     

Thermal acclimation of metabolic rate may be seasonally dependent in the subtropical anuran Latouche's frog (Rana latouchii, Boulenger)

We tested the hypothesis that the lack of metabolic thermal acclimation ability in tropical and subtropical amphibians is dependent on season and investigated the effects of body size, sex, time of day, and season on metabolic rates in Rana latouchii. The males were acclimated at 15 degrees, 20 degrees, and 25 degrees C, and their oxygen consumption was measured at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C in all four seasons, with the exception that we did not measure oxygen consumption at 30 degrees C in winter frogs. We also acclimated the males at 30 degrees C in summer for investigating diel variation of metabolic rate. The females were acclimated at 20 degrees and 25 degrees C, and their oxygen consumption was measured at 15 degrees , 20 degrees , 25 degrees , and 30 degrees C in summer. Our results showed that metabolic rates of R. latouchii differed by time of day, season, and acclimation temperature but did not differ by sex if the results were adjusted for differences in body mass. Summer males exhibited a 26%-48% increase in metabolic rates from the lowest values in the seasons. There was a trend of increased oxygen consumption in cold-acclimated males, but it was significant only at 15 degrees and 25 degrees C in summer, autumn, and winter. These results support the hypothesis that thermal acclimation of metabolism is seasonally dependent, which has not been reported in other tropical and subtropical amphibians.     

Comparative chlorine and temperature tolerance of the oyster Crassostrea madrasensis: implications for cooling system fouling

Crassostrea madrasensis is an important fouling oyster in tropical industrial cooling water systems. C. madrasensis individuals attach to surfaces by cementing one of their two valves to the substratum. Therefore, oyster fouling creates more problems than mussel fouling in the cooling conduits of power stations, because unlike the latter, the shell of the former remains attached to the substratum even after the death of the animal. However, there are no published reports on the tolerance of this species to chlorination and heat treatment. The mortality pattern and physiological behaviour (oxygen consumption and filtration rate) of three size groups (13 mm, 44 mm and 64 mm mean shell length) of C. madrasensis were studied at different residual chlorine concentrations (0.25, 0.5, 0.75, 1, 2, 3 to 5 mg 1-1) and temperatures (30 degrees C to 45 degrees C). The effect of shell size (= age) on C. madrasensis mortality in the presence of chlorine and taking into account temperature was significant, with the largest size group oysters showing highest resistance. At 1 mg l-1 residual chlorine, the 13 mm and 64 mm size group oysters, took 504 h (21 d) and 744 h (31 d), respectively to reach 100% mortality. At 39 degrees C, the 13 mm size group oysters took 218 min to reach 100% mortality, whereas the 64 mm size group oysters took 325 min. The oxygen consumption and filtration rate of C. madrasensis showed progressive reduction with increasing residual chlorine concentrations. However, the filtration rate and oxygen consumption responses of C. madrasensis were not significantly different between 30 degrees C (control) and 37.5 degrees C. There was a sharp decrease in the filtration rate and oxygen consumption at 38.5 degrees C. A comparison of the present mortality data with previous reports on other bivalves suggests that the chlorine tolerance of C. madrasensis lies in between that of Perna viridis and Perna perna, while its temperature tolerance is significantly higher than that of the other two bivalve species. However, in power station heat exchangers, where simultaneous chlorine and thermal stresses are existent, C. madrasensis may have an edge over other common foulants, because of its high temperature tolerance.     

Effect of hypercapnia on thermoregulation at high ambient temperature

The reported investigations were carried out on rabbits exposed for three hours to ambient temperature of 25 degrees C or 35 degrees breathing athmospheric air (controls) or gas mixtures containing 4% or 7% of CO2. During the exposure to 35 degrees C in rabbits breathing the gas mixture with 7% of CO2 the rise of rectal temperature was significantly greater, heat elimination from the auricular surface was increased, whereas the oxygen uptake was increased insignificantly. In tracheostomized rabbits breathing the gas mixture with 7% of CO2 at 32 degrees C the respiratory rate decreased but the respiration volume increased as compared with the animals breathing atmospheric air. It seems that the hyperthermic effect of hypercapnia demonstrated in this work can be attributed to the impairment of heat elimination through the upper airways due to an inhibition of thermal panting.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.