How to measure the thermal death of Daphnia? A comparison of different heat tests and effects of heat injury

1. 1. The thermal death point of the water flea Daphnia magna (age < 24 h, cultured at 20°C) varied considerably depending on the method used. The median lethal dose (LD₅₀), induced by an acute 24 h heat exposure was 34.8°C. It was 37.8°C following a thermal shock for 15 min, and it was 39.4°C when a continuous temperature increase (0.2°C/min) was used.

2. 2. Heat death temperature of daphnids was related to the acute heating rate.

3. 3. The logarithm of median lethal time (Lt₅₀) of daphnids, kept at a constant high temperature, had a linear relationship to temperature (°C) within the range of 28.0–38.5°C.

4. 4. The mortality after heat exposure increased with recovery time at 20°C for up to 3 days.

5. 5. The animals which survived the heat exposure produced eggs and offspring. Furthermore, no time lag in development between the control and heat exposure group was observed.

6. 6. The comparison of the results made by different heat tests categorized to Methods 1 and 2 by Precht (1973), for use in the determination of lethal limits of ectotherms, has been discussed.

     

Reversible and irreversible modifications ofβ-lactoglobulin upon exposure to heat

Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium ofβ-lactoglobulin upon thermal treatment at neutralpH were studied. Exposure of tryptophan residues was temperature dependent and was essentially completed on the time scale of seconds. Reorganization of generic hydrophobic protein patches on the protein surface was monitored through binding of 1,8-anilinonaphthalenesulfonate, and was much slower than changes in tryptophan exposure. Different phases in surface hydrophobicity changes were related to the swelling and the subsequent collapse of the protein, which formed a metastable swollen intermediate. Heat treatment ofβ-lactoglobulin also resulted in the formation of soluble oligomeric aggregates. The aggregation process was studied as a function of temperature, demonstrating that (i) dimer dissociation was a necessary step in a sequential polymerization mechanism and (ii) cohesion of hydrophobic patches was the major driving force for aggregation.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (Re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed. © 1996 Wiley-Liss, Inc.     

Why do cells require heat shock proteins to survive heat stress?

The cellular response to heat stress includes the induction of a group of proteins called the Heat Shock Proteins, whose functions include the synthesis of the thermoprotectant trehalose, refolding of denatured proteins, and ubiquitin- and proteasome-dependent degradation. Recent studies show that simply increasing the activity of ubiquitin- and proteasome-dependent degradation can replace the essential functions played by the induction of heat shock proteins during a heat stress. These results suggest that accumulation of denatured or aggregated proteins is the reason for the loss of cell viability due to heat stress.     

The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat–exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T _c). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete’s tapering period for the 2007 Marathon Des Sables. The subject (VO₂max = 50.7 ml·kg⁻¹·min⁻¹, peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO₂max in a temperature-controlled room (average WBGT = 31.9 ± 0.9°C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T _c, heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T _c and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman’s rank correlation = −0.81, p < 0.01). Furthermore, the 15-day heat–exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

Growth stimulating activity of heat shock protein 90α to lymphoid cell lines in serum-free medium

A growth stimulating factor was purified from the culture supernatant of human-human hybridoma SH-76 cells in serum-free RPMI 1640 medium by the serial use of DEAE anion and heparin affinity chromatographies. The factor was a 85 kDa protein on SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence (PEETQTQDQPME) of the protein was coincident with that of heat shock protein 90α (HSP90α). The protein reacted with anti-HSP90 monoclonal antibody. These results suggest that the protein was a member of HSP90α family after taking other circumstantial evidence into account. The protein stimulated the growth of some lymphoid cell lines such as human B-lymphoblastoid cell line HO-323-3 hybridomas derived from HO-323, and several other lymphoid cell lines. There were several lymphoid cell lines which did not respond to the protein. Growth stimulating activity of the protein was heat-unstable and significantly decreased at above 60°C. These are the first data that describe growth-stimulating activity of heat shock protein 90α.     

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.     

The heat dissipation limit theory and evolution of life histories in endotherms--time to dispose of the disposable soma theory?

A major factor influencing life-history strategies of endotherms is body size. Larger endotherms live longer, develop more slowly, breed later and less frequently, and have fewer offspring per attempt at breeding. The classical evolutionary explanation for this pattern is that smaller animals experience greater extrinsic mortality, which favors early reproduction at high intensity. This leads to a short lifespan and early senescence by three suggested mechanisms. First, detrimental late-acting mutations cannot be removed because of the low force of selection upon older animals (mutation accumulation). Second, genes that promote early reproduction will be favored in small animals, even if they have later detrimental effects (antagonistic pleiotropy). Third, small animals may be forced to reduce their investment in longevity assurance mechanisms (LAMs) in favor of investment in reproduction (the disposable soma theory, DST). The DST hinges on three premises: that LAMs exist, that such LAMs are energetically expensive and that the supply of energy is limited. By contrast, the heat dissipation limit (HDL) theory provides a different conceptual perspective on the evolution of life histories in relation to body size. We suggest that rather than being limited, energy supplies in the environment are often unlimited, particularly when animals are breeding, and that animals are instead constrained by their maximum capacity to dissipate body heat, generated as a by-product of their metabolism. Because heat loss is fundamentally a surface-based phenomenon, the low surface-to-volume ratio of larger animals generates significant problems for dissipating the body heat associated with reproductive effort, which then limits their current reproductive investment. We suggest that this is the primary reason why fecundity declines as animal size increases. Because large animals are constrained by their capacity for heat dissipation, they have low reproductive rates. Consequently, only those large animals living in habitats with low extrinsic mortality could survive leading to the familiar patterns of life-history trade-offs and their links to extrinsic mortality rates. The HDL theory provides a novel mechanism underpinning the evolution of life history and ageing in endotherms, and makes a number of testable predictions that directly contrast with the predictions arising from the DST.     

Sows’ responses to increased heat load – A review

There is a comprehensive body of literature on how increased air temperature affects the physiology, production and behaviour of sows, while very few studies consider the thermal effects of air humidity and air velocity.This review summarises studies that have investigated effects of air temperature by reviewing published literature in which sows were exposed to at least two different levels of air temperature ranging from 15 °C to 39 °C. Increased rectal temperature was investigated in the majority of the studies (26) and on average, the rectal temperature increased by 0.099 °C per °C increased air temperature above 25 °C. The increase was smaller at lower air temperatures, and it was suggested that rectal temperature is practically unaffected by air temperatures in the range of 15 °C–21 °C. This review elucidates how air temperature also affects performance indicators such as respiration rate, vaginal temperature, skin temperature, feed intake, milk yield, body weight loss during lactation, mortality, litter daily weight gain during lactation and sow behaviour.One study reported how respiration rate, rectal temperature, vaginal temperature and skin temperature were affected by both air temperature and air humidity, and the results suggest that the relative significance of air temperature and humidity may be similar for sows and finishing pigs (e.g. an increase of 40% relative humidity at an air temperature of 30 °C has a similar effect as a 1.9 °C increase in temperature).Studies on mitigation methods against the effects of high temperature and humidity such as snout cooling, drip cooling and floor cooling were reviewed to extract knowledge related to the effects of air velocity, temperatures of surrounding surfaces and the opportunity for sows to moisten their skin.     

β-Alanine suppresses heat inactivation of lactate dehydrogenase

β-Alanine exhibits neurotransmitter activity and is a component of the anti-glycation agent carnosine. We propose that β-alanine may have additional properties which may be of physiological significance. Interestingly, stress modulates the level of β-alanine, which regulates excitotoxicity responses and prevents neuronal cell death. We hypothesize that β-alanine's protective role may involve preservation of enzyme structure and function, suggesting that β-alanine may act as a chemical chaperone. We used light scattering, enzyme activity and intrinsic fluorescence to monitor heat-induced changes in lactate dehydrogenase (LDH) in the presence and absence of β-alanine. We observed that β-alanine suppressed heat-induced LDH inactivation, prevented LDH aggregation, ameliorated the decrease in intrinsic fluorescence and reactivated thermally denatured LDH. These observations support the hypothesis that β-alanine has chaperone-like activity and may play a cellular role in the preservation of enzyme function.     

Physiological response to heat stress of tomato ‘Micro-Tom’ plants expressing high and low levels of mitochondrial sHSP23.6 protein

Tomato ‘Micro-Tom’ plants were transformed for high or low expression of the mitochondrial small “heat shock” protein (HSP) (MT-sHSP23.6) to evaluate their response to high temperature. The plants were raised for 59 days under a controlled temperature, photoperiod and photon flow density and then subjected to heat stress for 24 h at 37 °C, followed by a recovery period under normal conditions (21 ± 2 °C). The cycle was repeated. The chlorophyll a fluorescence intensity was measured, and the parameters of the JIP-test were calculated. The gas exchange was also evaluated. The JIP-test showed significantly different responses of the genotypes to heat stress. The parameters of photosystem I activity and the net assimilation of CO₂ increased during the first stress cycle in genotypes with a high expression of MT-sHSP23.6 and in non-transformed plants; however, the net assimilation of CO₂ decreased in genotypes with a low expression of MT-sHSP23.6. The data suggest that MT-sHSP23.6 participates in the heat tolerance mechanism, considering that the suppression of this protein resulted in greater physiological damage during heat stress.     

Impact of heat stress on conception rate of dairy cows in the moderate climate considering different temperature–humidity index thresholds,periods relative to breeding,and heat load indices

The objectives of this retrospective study were to investigate the relationship between temperature–humidity index (THI) and conception rate (CR) of lactating dairy cows, to estimate a threshold for this relationship, and to identify periods of exposure to heat stress relative to breeding in an area of moderate climate. In addition, we compared three different heat load indices related to CR: mean THI, maximum THI, and number of hours above the mean THI threshold. The THI threshold for the influence of heat stress on CR was 73. It was statistically chosen based on the observed relationship between the mean THI at the day of breeding and the resulting CR. Negative effects of heat stress, however, were already apparent at lower levels of THI, and 1 hour of mean THI of 73 or more decreased the CR significantly. The CR of lactating dairy cows was negatively affected by heat stress both before and after the day of breeding. The greatest negative impact of heat stress on CR was observed 21 to 1 day before breeding. When the mean THI was 73 or more in this period, CR decreased from 31% to 12%. Compared with the average maximum THI and the total number of hours above a threshold of more than or 9 hours, the mean THI was the most sensitive heat load index relating to CR. These results indicate that the CR of dairy cows raised in the moderate climates is highly affected by heat stress.     

Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone–estradiol-based protocols

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone–estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 – 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 – no chalk removal=no estrus expression; HEATSC2 – partial chalk removal=low estrus expression; HEATSC3 – near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P<0.05). In Exp. 2, P/AI (HEATSC1 – 40.0%; HEATSC2 – 49.7%; HEATSC3 – 60.9%; P<0.001), and larger follicle timed artificial insemination (LFTAI) (<0.001) varied according to HEATSC. There was no difference in P/AI (P=0.41) or LFTAI (P=0.33) according to ovulation inducer. Cows with greater BCS showed greater P/AI in both experiments (P<0.05). Estrus presence and greater HEATSC improved P/AI, and EC v. EB used promoted differential estrus manifestation (cows showing HEATSC2 and HEATSC3: 79.5% with EB v. 69.98% with EC use, P<0.001), however, with similar P/AI. The use of HEATSC in B. indicus cows subjected to TAI is useful to identify cows with greater estrus expression and consequently improved pregnancy rates in TAI, allowing the cows with low HEATSC to be targeted for additional treatments aimed at improving P/AI.     

A comparison of two fouling‐resistant heat exchangers

Fouling cannot always be prevented; it is important to consider the design of fouling‐resistant heat exchangers. To examine these exchangers, a test fluid whose fouling behaviour is understood should be used. Experiments have been conducted to examine the response of two model systems, a pulsatile flow and a fluid bed heat exchanger, to fouling from whey protein concentrates. Both systems are effective in certain cases, although the enhanced mass transfer possible in the pulsatile flow exchanger can increase fouling when mass transfer controls deposition. This demonstrates the possible danger in installing “antifouling”; systems. The possible mechanisms by which antifouling exchangers operate is discussed; they may work both by slowing the kinetics of fouling or enhancing the heat transfer coefficient. A simple model to demonstrate the design of antifouling exchangers is presented.     

Phase separation—a strategy to resist autophagic degradation under heat stress

<正>Stress spans our whole lives. When exposed to stressful conditions, every life, from a worm to a human, adjusts its internal properties to adapt to new environments. This principle applies to adults, and is also essential during development in animals. Dr. Zhang and coworkers from Institute of Biophysics, Chinese Academy of Sciences made a remarkable discovery showing that heat-stress-triggered accumulation of PGL granules is an adaptive response to maintain embryonic viability (Figure 1). Their work was published in the September 6th issue of Cell, entitled"mTOR regulates phase separation of PGL granules to modulate their autophagic degradation"(Zhang et al., 2018).     

Identification of chilling and heat requirements of cherry trees—a statistical approach

Most trees from temperate climates require the accumulation of winter chill and subsequent heat during their dormant phase to resume growth and initiate flowering in the following spring. Global warming could reduce chill and hence hamper the cultivation of high-chill species such as cherries. Yet determining chilling and heat requirements requires large-scale controlled-forcing experiments, and estimates are thus often unavailable. Where long-term phenology datasets exist, partial least squares (PLS) regression can be used as an alternative, to determine climatic requirements statistically. Bloom dates of cherry cv. ‘Schneiders späte Knorpelkirsche’ trees in Klein-Altendorf, Germany, from 24 growing seasons were correlated with 11-day running means of daily mean temperature. Based on the output of the PLS regression, five candidate chilling periods ranging in length from 17 to 102 days, and one forcing phase of 66 days were delineated. Among three common chill models used to quantify chill, the Dynamic Model showed the lowest variation in chill, indicating that it may be more accurate than the Utah and Chilling Hours Models. Based on the longest candidate chilling phase with the earliest starting date, cv. ‘Schneiders späte Knorpelkirsche’ cherries at Bonn exhibited a chilling requirement of 68.6?±?5.7 chill portions (or 1,375?±?178 chilling hours or 1,410?±?238 Utah chill units) and a heat requirement of 3,473?±?1,236 growing degree hours. Closer investigation of the distinct chilling phases detected by PLS regression could contribute to our understanding of dormancy processes and thus help fruit and nut growers identify suitable tree cultivars for a future in which static climatic conditions can no longer be assumed. All procedures used in this study were bundled in an R package (‘chillR’) and are provided as Supplementary materials. The procedure was also applied to leaf emergence dates of walnut (cv. ‘Payne’) at Davis, California.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.