EXPERIMENTAL EVOLUTION OF HSP70 EXPRESSION AND THERMOTOLERANCE IN DROSOPHILA MELANOGASTER

To examine whether recent evolutionary history affects the expression of Hsp70, the major heat-induced-heat shock protein in Drosophila melanogaster, we measured Hsp70 expression, thermotolerance, and hsp70 gene number in replicate populations undergoing laboratory evolution at different temperatures. Despite Hsp70's ancient and highly conserved nature, experimental evolution effectively and replicably modified its expression and phenotype (thermotolerance). Among five D. melanogaster populations founded from a common ancestral population and raised at three different temperatures (one at 18°C, two each at 25°C and 28°C) for twenty years, Hsp70 expression varies in a consistent pattern: the replicate 28°C lines expressed 30–50% less Hsp70 than the other lines at a range of inducing temperatures. This modification was refractory to acclimation, and correlated with thermotolerance: the 28°C lines had significantly lower inducible tolerance of 38.5°C and 39°C. We verified the presence of five hsp70 genes in the genome of each line, excluding copy number variation as a candidate molecular basis of the evolved difference in expression. These findings support the ability of Hsp70 levels in D. melanogaster populations to change over microevolutionary time scales and implicate constancy of environmental temperature as a potentially important selective agent.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Thermotolerance of the photosynthetic light reactions in two <Emphasis Type="Italic">Phaseolus</Emphasis> species: a comparative study

The common bean (Phaseolus vulgaris L.) is sensitive to high temperature, while an ecologically contrasting species (Phaseolus acutifolius A. Gray) is cultivated successfully in hot environments. In this study, the two bean species were respectively acclimated to a control temperature of 25 °C and a moderately elevated temperature of 35 °C in order to compare the thermotolerance capabilities of their photosynthetic light reactions. Growth at 35 °C appeared to have no obvious adverse effect on the photosynthetic activities of the two beans, but changed their thermotolerance. After a short period of heat shock (40 °C for up to 4 h), the photosynthetic activities of 25 °C-grown P. vulgaris declined more severely than those of P. acutifolius grown at 25 °C, implying that the basal thermotolerance of P. vulgaris is inferior to that of P. acutifolius. But after acclimating to 35 °C, the thermotolerances of the two species were both greatly enhanced to about the same level, clearly demonstrating the induction of acquired thermotolerance in their chloroplasts, and P. vulgaris could be as good as P. acutifolius. Temperature acclimation also changed plants’ resistance to photoinhibition in a manner similar to those toward heat stress. In addition, acquisition of tolerance to heat and strong irradiance would reduce the dependency of the two beans on xanthophyll pigments to dissipate heat, and also seemed irrelevant to the agents with antioxidant activities such as SOD.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Plastic and evolutionary responses to heat stress in a temperate dung fly: negative correlation between basal and induced heat tolerance?

Extreme weather events such as heat waves are becoming more frequent and intense. Populations can cope with elevated heat stress by evolving higher basal heat tolerance (evolutionary response) and/or stronger induced heat tolerance (plastic response). However, there is ongoing debate about whether basal and induced heat tolerance are negatively correlated and whether adaptive potential in heat tolerance is sufficient under ongoing climate warming. To evaluate the evolutionary potential of basal and induced heat tolerance, we performed experimental evolution on a temperate source population of the dung fly Sepsis punctum. Offspring of flies adapted to three thermal selection regimes (Hot, Cold and Reference) were subjected to acute heat stress after having been exposed to either a hot‐acclimation or non‐acclimation pretreatment. As different traits may respond differently to temperature stress, several physiological and life history traits were assessed. Condition dependence of the response was evaluated by exposing juveniles to different levels of developmental (food restriction/rearing density) stress. Heat knockdown times were highest, whereas acclimation effects were lowest in the Hot selection regime, indicating a negative association between basal and induced heat tolerance. However, survival, adult longevity, fecundity and fertility did not show such a pattern. Acclimation had positive effects in heat‐shocked flies, but in the absence of heat stress hot‐acclimated flies had reduced life spans relative to non‐acclimated ones, thereby revealing a potential cost of acclimation. Moreover, body size positively affected heat tolerance and unstressed individuals were less prone to heat stress than stressed flies, offering support for energetic costs associated with heat tolerance. Overall, our results indicate that heat tolerance of temperate insects can evolve under rising temperatures, but this response could be limited by a negative relationship between basal and induced thermotolerance, and may involve some but not other fitness‐related traits.     

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.     

A comprehensive assessment of geographic variation in heat tolerance and hardening capacity in populations of Drosophila melanogaster from eastern Australia

We examined latitudinal variation in adult and larval heat tolerance in Drosophila melanogaster from eastern Australia. Adults were assessed using static and ramping assays. Basal and hardened static heat knockdown time showed significant linear clines; heat tolerance increased towards the tropics, particularly for hardened flies, suggesting that tropical populations have a greater hardening response. A similar pattern was evident for ramping heat knockdown time at 0.06 °C min^?1 increase. There was no cline for ramping heat knockdown temperature (CT_max) at 0.1 °C min^?1 increase. Acute (static) heat knockdown temperature increased towards temperate latitudes, probably reflecting a greater capacity of temperate flies to withstand sudden temperature increases during summer in temperate Australia. Larval viability showed a quadratic association with latitude under heat stress. Thus, patterns of heat resistance depend on assay methods. Genetic correlations in thermotolerance across life stages and evolutionary potential for critical thermal limits should be the focus of future studies.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Effects of acclimation and latitude on the activity thresholds of the aphid Myzus persicae in Europe

Activity thresholds were measured in nine anholocyclic clones of the peach‐potato aphid Myzus persicae collected along a latitudinal cline of its European distribution from Sweden to Spain. The effects of collection origin and intra‐ and intergenerational acclimation on these thresholds were investigated. Low‐temperature (10°C) acclimation for one generation depressed the movement threshold and chill coma temperatures, with the largest reduction in movement threshold recorded for clone UK 1 (8.8–2.5°C) and in chill coma for UK 2 (4.8–2.0°C). High‐temperature (25°C) acclimation for one generation increased the heat movement threshold and heat coma temperature with the largest increase in the movement threshold (40.1–41.1°C) and heat coma (41.4–42.3°C) recorded for clone Swed 1. There was no further intergenerational acclimation over three generations. High‐temperature activity thresholds were less plastic than low‐temperature thresholds, and, consequently, thermal activity ranges were expanded following low‐temperature acclimation. No constant affect of acclimation was observed on chill coma recovery, although clonal differences were observed with Swed 1 and 3 requiring some of the longest complete recovery times. There was no relationship between latitude and activity thresholds with the exception of heat coma data where Scandinavian clones Swed 2 and 3 consistently displayed some of the lowest heat coma temperatures (e.g. 41.3°C for both clones at 20°C) and Mediterranean clones Span 1, 2 and 3 displayed some of the highest (e.g. 42.1, 41.9 and 42.5°C, respectively, at 20°C). These data suggest that clonal mixing could occur over a large scale across Europe, limiting local adaptation to areas where conditions enable long‐term persistence of populations, e.g. adaptation to higher temperatures in the Mediterranean region. It is suggested that aphid thermal tolerance could be governed more by clonal type than the latitudinal origin.     

Strong responses of Drosophila melanogaster microbiota to developmental temperature

Physiological responses to changes in environmental conditions such as temperature may partly arise from the resident microbial community that integrates a wide range of bio-physiological aspects of the host. In the present study, we assessed the effect of developmental temperature on the thermal tolerance and microbial community of Drosophila melanogaster. We also developed a bacterial transplantation protocol in order to examine the possibility of reshaping the host bacterial composition and assessed its influence on the thermotolerance phenotype. We found that the temperature during development affected thermal tolerance and the microbial composition of male D. melanogaster. Flies that developed at low temperature (13°C) were the most cold resistant and showed the highest abundance of Wolbachia, while flies that developed at high temperature (31°C) were the most heat tolerant and had the highest abundance of Acetobacter. In addition, feeding newly eclosed flies with bacterial suspensions from intestines of flies developed at low temperatures changed the heat tolerance of recipient flies. However, we were not able to link this directly to a change in the host bacterial composition.     

Low night temperatures have a differential effect on the diurnal cycling of gene expression in cold–sensitive and tolerant tomatoes*

Abstract. Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold–sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly(A)⁺ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulation of six to eight mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response.     

Effect of latitude and acclimation on the lethal temperatures of the peach‐potato aphid Myzus persicae

• 1 Aphids, similar to all insects, are ectothermic and, consequently, are greatly affected by environmental conditions. The peach potato aphid Myzus persicae (Sulzer) has a global distribution, although it is not known whether populations display regional adaptations to distinct climatic zones along its distribution and vary in their ability to withstand and acclimate to temperature extremes. In the present study, lethal temperatures were measured in nine anholocyclic clones of M. persicae collected along a latitudinal cline of its European distribution from Sweden to Spain. The effects of collection origin and intra‐ and intergenerational acclimation on cold and heat tolerance, as determined by upper and lower lethal temperatures (ULT₅₀ and LLT₅₀, respectively), were investigated.
• 2 Lethal temperatures of M. persicae were shown to be plastic and could be altered after acclimation over just one generation. Lower lethal temperatures were significantly depressed in eight of nine clones after acclimation for one generation at 10°C (range: ?13.3 to ?16.2°C) and raised after acclimation at 25°C (range: ?10.7 to ?11.6°C) compared with constant 20°C (range: ?11.9 to ?12.9°C). Upper lethal temperatures were less plastic, although significantly increased after one generation at 25°C (range: 41.8–42.4°C) and in five of nine clones after acclimation at 10°C. There was no evidence of intergenerational acclimation over three generations.
• 3 Thermal tolerance ranges were expanded after acclimation at 10 and 25°C compared with constant 20°C, resulting in aphids reared at 10°C surviving over a temperature range that was approximately 2–6°C greater than those reared at 25°C.
• 4 There was no clear relationship between lethal temperatures and latitude. Large scale mixing of clones may occur across Europe, thus limiting local adaption in thermal tolerance. Clonal type, as identified by microsatellite analysis, did show a relationship with thermal tolerance, notably with Type O clones being the most thermal tolerant. Clonal types may respond independently to climate change, affecting the relative proportions of clones within populations, with consequent implications for biodiversity and agriculture.

     

Within‐generation variation of critical thermal limits in adult Mediterranean and Natal fruit flies Ceratitis capitata and Ceratitis rosa: thermal history affects short‐term responses to temperature

Insect thermal tolerance shows a range of responses to thermal history depending on the duration and severity of exposure. However, few studies have investigated these effects under relatively modest temperature variation or the interactions between short‐ and longer‐term exposures. In the present study, using a full‐factorial design, 1 week‐long acclimation responses of critical thermal minimum (CT_min) and critical thermal maximum (CT_max) to temperatures of 20, 25 and 30 °C are investigated, as well as their interactions with short‐term (2 h) sub‐lethal temperature exposures to these same conditions (20, 25 and 30 °C), in two fruit fly species Ceratitis capitata (Wiedemann) and Ceratitis rosa Karsch from South Africa. Flies generally improve heat tolerance with high temperature acclimation and resist low temperatures better after acclimation to cooler conditions. However, in several cases, significant interaction effects are evident for CT_max and CT_min between short‐ and long‐term temperature treatments. Furthermore, to better comprehend the flies' responses to natural microclimate conditions, the effects of variation in heating and cooling rates on CT_max and CT_min are explored. Slower heating rates result in higher CT_max, whereas slower cooling rates elicit lower CT_min, although more variation is detected in CT_min than in CT_max (approximately 1.2 versus 0.5 °C). Critical thermal limits estimated under conditions that most closely approximate natural diurnal temperature fluctuations (rate: 0.06 °C min^?1) indicate a CT_max of approximately 42 °C and a CT_min of approximately 6 °C for these species in the wild, although some variation between these species has been found previously in CT_max. In conclusion, the results suggest critical thermal limits of adult fruit flies are moderated by temperature variation at both short and long time scales and may comprise both reversible and irreversible components.     

Arabidopsis: Sensitivity of growth to high temperature

Arabidopsis thaliana seedlings as measured by an electrolyte leakage assay, have been found to be extremely sensitive to high temperature stress as compared to a high temperature tolerant variety (Tracy) of soybean. Over 50% ion leakage occurred in Arabidopsis leaves during a 15-minute exposure to 50°C, indicating a heat killing time of less than 15 minutes. In contrast, the heat killing time for soybean at 50°C was over five times longer. When soybean or Arabidopsis seedlings in culture plates were exposed to 37°C for 2 hours and then returned to 23°C, they suffered no apparent short-term or long-term damage. Soybean seedlings given a 42°C, treatment for 2 hours also showed no damage. Arabidopsis seedlings after a 42°C treatment for 2 hours showed no apparent immediate damage, but 48 hours after return to 23°C severe damage symptoms were visible and after 96 hours all the seedlings were dead. Both soybean and Arabidopsis seedlings synthesize heat shock proteins (hsps) when exposed to 42°C for 2 hours. The hsps synthesized are of similar molecular weights, although the relative abundances of the different size classes are very different in the two plants. Even though hsps are produced in Arabidopsis seedlings after a 2 hour exposure to 42°C their presence is not sufficient for the seedlings to recover from the effects of rhe heat shock when returned to 23°C. Our results show that Arabidopsis has a heat sensitive genotype. This along with its other characteristics should make it a good model system in which to assay in transgenic plants, the functions of homologous and heterologous genes that might be candidates for determining heat tolerance in plants.     

Acclimation of different strains of the olive fly, Dacus oleae, to low temperatures

Male and female D. oleae have similar powers of acclimation when exposed to low temperatures. Their torpor thresholds depend upon the temperature to which they have been acclimatised. During slow cooling (i.e. less than 1°C per min) they are capable of some rapid acclimation which enables them to lower their torpor threshold by almost 1°C degree, as compared with when they are chilled quickly. After abrupt transfer from 25°C to a different temperature, acclimation takes some time to be accomplished. At 15°C and above it occurs within 10 days but at temperatures below this, progressive acclimation lowers the torpor thresholds to the very low levels typical of flies overwintering under natural conditions. During this long term acclimation torpor thresholds may change by almost 0.5°C per 1°C change of acclimation temperature.No differences were observed in the ability of either flies from northern and southern Greece, or normal and γ-irradiated laboratory reared flies to acclimate to winter conditions in the field. In all cases, torpor thresholds were progressively lowered in advance of the decline in weekly minimum temperatures.     

Involvement of nitric oxide in acquired thermotolerance of rice seedlings

The role of nitric oxide (NO) in thermotolerance acquired by heat acclimation (38°C) was investigated. Results showed that 38°C acclimation, on the one hand, obviously reduced hydrogen peroxide (H₂O₂) and MDA contents and ion leakage degree in rice leaves; however, on the other hand, it increased the survival of rice (Oryza sativa L.) seedlings under 50°C heat stress. Application of nitric oxide donor, sodium nitroprusside (SNP), prior to 38°C acclimation dramatically increased the acquired thermotolerance. To elucidate the role of endogenous NO in acquired thermotolerance, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a specific NO scavenger) was used (scavengers are used to control the level of both exogenous and endogenous NO). Results showed that PTIO pretreatment resulted in the elimination of acquired thermotolerance induced by 38°C acclimation in rice seedlings. Nitric oxide (NO) release measurement indicated that there was indeed an abrupt elevation in the NO content in 40 min after 38°C acclimation, proving the involvement of NO in acquired thermotolerance inducement in rice seedling.     

Prosopis chilensis is a plant highly tolerant to heat shock

At temperatures between 25 and 35°C, 100% of Prosopis chilensis seeds germinated within 24 h. At higher temperatures, the germination rate was reduced; at 50°C, seeds did not germinate. After germination at 25°C, the optimal temperature for seedling growth was 35°C and the seedlings did not grow at a temperature of 50°C. However, when germination was at 35°C, the optimal temperature for seedling growth was 40°C and some seedlings grew at 50°C, suggesting that thermotolerance was induced during seed germination at 35°C. Further thermotolerance can be induced in seedlings germinated at 35°C, by exposing them to 40°C for 2h. Under these conditions, seedlings exhibited increased growth rate at 45 and 50°C. Fluorography of SDS-polyacrylamide gel electrophoresis of the proteins synthesized and accumulated during 2 h at temperatures of 35, 40, 45 and 50°C in the presence of [³⁵S]methionine revealed the expression of 11 proteins not detectable at 35°C. Most of the proteins present at 35°C also increased in expression. The temperature for maximal expression of these proteins was 45°C.