The effect of thermal stress on the mating behavior of three Drosophila species.

Selection may act on the weakest link in fitness to change how a species adapts to an environmental stress. For many species, this limitation may be reproduction. After adult Drosophila melanogaster, Drosophila simulans, and Drosophila mojavensis males were exposed to varying levels of thermal stress well below those that endanger life, courtship and mating frequency declined. The regression coefficients of both courtship and mating success did not differ significantly between D. melanogaster and D. simulans males. In contrast, significant differences were present between the two cosmopolitan species and D. mojavensis. Courtship frequency decreased at a much slower rate in D. mojavensis than in D. melanogaster and D. simulans, and while heat-stressed D. mojavensis males continued to court, many did not mate. In the cosmopolitan species, courting males almost always mated successfully. Courtship behaviors, including wing waving, were observed in D. mojavensis at temperatures that prohibited flight, while flight, courtship, and mating were knocked out simultaneously in D. melanogaster. One possible explanation for decreased flight ability and courtship success may be the reduced heat shock response in the flight muscle tissue because Hsp70 expression was lowest in the thoracic tissue of both D. melanogaster and D. mojavensis.

Reduced enzyme activity following Hsp70 overexpression in Drosophila melanogaster.

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.

Changes in thermotolerance and Hsp70 expression with domestication in Drosophila melanogaster.

To examine how the duration of laboratory domestication may affect Drosophila stocks used in studies of thermotolerance, we measured expression of the inducible heat-shock protein Hsp70 and survival after heat shock in D. melanogaster strains recently collected from nature and maintained in laboratory culture for up to 50 or more generations. After an initial increase in both Hsp70 expression and thermotolerance immediately after transfer to laboratory medium, both traits remained fairly constant over time and variation among strains persisted through laboratory domestication. Furthermore, variation in heat tolerance and Hsp70 expression did not correlate with the length of time populations evolved in the laboratory. Therefore, while environmental variation likely contributed most to early shifts in strain tolerance and Hsp70 expression, other population parameters, for example genetic drift, inbreeding, and selection likely affected these traits little. As long as populations are maintained with large numbers of individuals, the culture of insects in the laboratory may have little effect on the tolerance of different strains to thermal stress.

A comparison of Hsp70 expression and thermotolerance in adults and larvae of three Drosophila species.

Heat shock proteins (Hsps) and other molecular chaperones perform diverse physiological roles. One is to facilitate, in part, organismal thermotolerance, of which the functional consequences depend on Hsp70 concentration and developmental stage in Drosophila melanogaster. To test whether an Hsp70-thermotolerance relationship is a general phenomenon within Drosophila, I assayed Hsp70 concentration at a range of temperatures in intact larvae and adults of three species, D. melanogaster, D. simulans, and D. mojavensis, and compared those results to the increase in survival to heat shock that occurs after an Hsp70 inducing pretreatment. Larvae of D. melanogaster and D. simulans responded similarly to heat; they expressed Hsp70 maximally at 36-37 degrees C, and their tolerance of 1 h heat shocks increased by 1.5-2 degrees C. By contrast, D. mojavensis, which tolerates higher temperatures than do D. melanogaster and D. simulans, expressed Hsp70 only at higher temperatures, although the 36 degrees C pretreatment still increased thermotolerance. Critically, the temperature that maximally induced Hsp70 was a poor inducer of thermotolerance in D. mojavensis and may have harmed larvae. Results for Drosophila adults, which tolerated heat poorly compared to larvae, likewise suggest that a close link between peak Hsp70 expression and maximal induction of thermotolerance is a feature of D. melanogaster, and not of the other species. Neither D. simulans nor D. mojavensis adults increased tolerance after exposure to the temperatures that maximally induced Hsp70.

A genetic analysis of the relationship between life-history variation and heat-shock tolerance in Drosophila buzzatii.

Although exposure to environmental stress is common in most populations, and the physiological effects of stress on individuals are well studied, the evolutionary importance of stress to populations is not well understood. To address multitrait responses to environmental change and potential constraints on character evolution, we analysed, in 100 isofemale lines of Drosophila buzzatii, the genetic relationships between resistance to a short heat shock and several life-history traits: survival in benign conditions, larval developmental time, fecundity and longevity. Estimates of heritability of larval thermotolerance were low, but significant, and all life-history traits varied significantly among isofemale lines. Several of these traits covaried significantly. Most correlations indicated positive life-history relationships, but males and females from lines where female fecundity was higher developed more slowly in the absence of stress, which is a negative life-history relationship. The stress reduced or negated many trait associations, and showed one additional relationship; more larvae from lines that developed fast at 25 degrees C survived to adult after stress than did larvae from slow developing lines. These shifts in fitness relationships, when a single stress bout is applied, suggest that even small increases in environmental stress can have profound effects on evolutionary relationships among life-history traits.

Hsp70 and larval thermotolerance in Drosophila melanogaster: how much is enough and when is more too much?

Heat shock proteins (Hsps) and other molecular chaperones perform diverse cellular roles (e.g., inducible thermotolerance) whose functional consequences are concentration dependent. We manipulated Hsp70 concentration quantitatively in intact larvae of Drosophila melanogaster to examine its effect on survival, developmental time and tissue damage after heat shock. Larvae of an extra-copy strain, which has 22 hsp70 copies, produced Hsp70 more rapidly and to higher concentrations than larvae of a control strain, which has the wild-type 10 copies of the gene. Increasing the magnitude and duration of pretreatment increased Hsp70 concentrations, improved tolerance of more severe stress, and reduced delays in development. Pretreatment, however, did not protect against acute tissue damage. For larvae provided a brief or mild intensity pretreatment, faster expression of Hsp70 in the extra-copy strain improved survival to adult and reduced tissue damage 21h after heat shock. Negative effects on survival ensued in extra-copy larvae pretreated most intensely, but their overexpression of Hsp70 did not increase tissue damage. Because rapid expression to yield a low Hsp70 concentration benefits larvae but overexpression harms them, natural selection may balance benefits and costs of high and low expression levels in natural populations.

Estimating heritability in a threshold trait: heat-shock tolerance in Drosophila buzzatii.

Stress tolerance is often measured as a threshold trait, the proportion of a group that survives a defined stress regime. Requirements of large offspring numbers coupled with fitness variation in the surviving cohort limit the use of some standard genetic analyses for estimating heritability. Therefore, we present an isofemale line analysis, which is a modified full-sib design, to estimate heritability of tolerance to heat shock in pretreated Drosophila buzzatii adults. Highly significant levels of genetic variation were found in males and females at the third generation of laboratory rearing, and the intraclass correlations were estimated to be about 0.2 for four independent sets of 25 isofemale lines. The proportion of the variance explained within lines among same-sex replicates, however, was larger than that between replicates of males and females. Because genetic variation was estimated from groups, the error variation required factoring by the group size to estimate heritability, which averaged 0.03. The four most tolerant, four least tolerant and four lines of average tolerance to heat stress in each set were reanalysed after 10-11 generations of rearing at 25 degrees C. Survival in the 13th-14th generations was positively and significantly associated with survival at generation 3. These comparisons indicate the high repeatability of measurements of heat-shock tolerance.

Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae.

All tissues of larval Drosophila melanogaster express Hsp70, the major heat-shock protein of this species, after both mild (36 degrees C) and severe (38.5 degrees C) heat shock. We used Hsp70-specific immunofluorescence to compare the rate and intensity of Hsp70 expression in various tissues after these two heat-shock treatments, and to compare this with related differences in the intensity of Trypan Blue staining shown by the tissues. Trypan Blue is a marker of tissue damage. Hsp70 was rarely detectable before heat shock. Brain, salivary glands, imaginal disks and hindgut expressed Hsp70 within the first hour of heat shock, whereas gut tissues, fat body and Malpighian tubules did not express Hsp70 until 4-21 h after heat shock. Differences in Hsp70 expression between tissues were more pronounced at the higher heat-shock temperature. Tissues that expressed Hsp70 slowly stained most intensely with Trypan Blue. Gut stained especially intensely, which suggests that its sensitivity to heat shock may limit larval thermotolerance. These patterns further suggest that some cells respond primarily to damage caused by heat shock rather than to elevated temperature per se and/or that Hsp70 expression is itself damaged by heat and requires time for recovery in some tissues.

Deleterious consequences of Hsp70 overexpression in Drosophila melanogaster larvae.

We compared transgenic Drosophila larvae varying in hsp70 copy number to assess the consequences of Hsp70 overexpression for growth and development after heat shock. Exposure to a mildly elevated temperature (36 degrees C) induced expression of Hsp70 (and presumably other heat shock proteins) and improved tolerance of more severe heat stress, 38.5-39.5 degrees C. We examined this pattern in two independently derived pairs of extra-copy and excision strains that differed primarily in hsp70 copy number (with 22 and 10 copies, respectively). Extra-copy larvae produced more Hsp70 in response to high temperature than did excision larvae, but surpassed the excision strain in survival only immediately after thermal stress. Excision larvae survived to adulthood at higher proportions than did extra-copy larvae and grew more rapidly after thermal stress. Furthermore, multiple pretreatment reduced survival of 1st-instar extra-copy larvae, but did not affect the corresponding excision strain. While extra Hsp70 provides additional protection against the immediate damage from heat stress, abnormally high concentrations can decrease growth, development and survival to adulthood.

Ecological and evolutionary physiology of heat shock proteins and the stress response in Drosophila: complementary insights from genetic engineering and natural variation.

Classical adaptational and genetic engineering approaches offer complementary insights to understanding biological variation: the former elucidates the origins, magnitude and ecological context of natural variation, while the latter establishes which genes can underlie natural variation. Studies of the stress or heat shock response in Drosophila illustrate this point. At the cellular level, heat shock proteins (Hsps) function as molecular chaperones, minimizing aggregation of peptides in non-native conformations. To understand the adaptive significance of Hsps, we have characterized thermal stress that Drosophila experience in nature, which can be substantial. We used these findings to design ecologically relevant experiments with engineered Drosophila strains generated by unequal site-specific homologous recombination; these strains differ in hsp70 copy number but share sites of transgene integration. hsp70 copy number markedly affects Hsp70 levels in intact Drosophila, and strains with extra hsp70 copies exhibit corresponding differences in inducible thermotolerance and reactivation of a key enzyme after thermal stress. Elevated Hsp70 levels, however, are not without penalty; these levels retard growth and increase mortality. Transgenic variation in hsp70 copy number has counterparts in nature: isofemale lines from nature vary significantly in Hsp70 expression, and this variation is also correlated with both inducible thermotolerance and mortality in the absence of stress.

High-temperature stress and the evolution of thermal resistance in Drosophila.

The evolution of thermal resistance and acclimation is reviewed at the population level using populations and isofemale lines of Drosophila buzzatii and D. melanogaster originating from different climatic regions. In general, ample genetic variation for thermal resistance was found within and among populations. A rough correlation between the climate of origin and thermal resistance was apparent. Acclimation at a non-lethal temperature led to a significant increase in survival after heat shock, and recurrent acclimation events generally increased survival even further. Acclimation effects lasted over several days, but this effect decreased gradually with time since acclimation. Protein studies showed that the concentration of Hsp70 in adult flies is greatly increased by acclimation and thereafter gradually decreases with time. For populations with relatively high survival at one life stage, survival often was low at other life stages. Furthermore, selection on different life stages showed that a selection response in one life stage did not necessarily result in a correlated response in another. These observations indicate that different mechanisms or genes at least in part are responsible for or are expressed at different developmental stages. Selection for increased resistance was successful despite low heritabilities for the trait. Survival and fertility were compared between acclimated and non-acclimated flies, and a cost of expressing the "heat shock response" was identified in that increased survival after acclimation was accompanied by reduced fertility. The relative costs increased under nutritional stress. Metabolic rate was genetically variable but did not correlate with temperature resistance. The more resistant lines, however, often had shorter developmental time. Inbreeding reduced thermal stress tolerance of adult flies, but it did not reduce tolerance of embryos that possibly are exposed to strong natural selection for thermal stress resistance. In general, inbreeding may reduce stress resistance, and thus multiple stressful events may account for increased inbreeding depression in harsh environments.

Effect of engineering Hsp70 copy number on Hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster.

To determine how the accumulation of the major Drosophila melanogaster heat-shock protein, Hsp70, affects inducible thermotolerance in larvae and pupae, we have compared two sister strains generated by site-specific homologus recombination. One strain carried 12 extra copies of the Hsp70 gene at a single insertion site (extra-copy strain) and the other carried remnants of the transgene construct but lacked the extra copies of Hsp70 (excision strain). Hsp70 levels in whole-body lysates of larvae and pupae were measured by ELISA with an Hsp70-specific antibody. In both extra-copy and excision strains, Hsp70 was undetectable prior to heat shock. Hsp70 concentrations were higher in the extra-copy strain than in the excision strain at most time points during and after heat shock. Pretreatment (i.e. exposure to 36 degrees C before heat shock) significantly improved thermotolerance, and this improvement was greater and more rapid in larvae and pupae of the extra-copy strain than in those of the excision strain. The experimental conditions resemble thermal regimes actually experienced by Drosophila in the field. Thus, these findings represent the best evidence to date that the amount of a heat-shock protein affects the fitness of a complex animal in the wild.

Acclimation and selection for increased resistance to thermal stress in Drosophila buzzatii.

Direct selection for increased resistance to a heat shock (41.9 degrees for 90 min) was carried out using two replicate lines of Drosophila buzzatii that were derived from a large base population. Selected individuals were first acclimated to high temperature before selection, while control individuals were acclimated but not selected, and selection was performed every second generation. Resistance to heat shock with acclimation increased in selected lines. Without acclimation, a correlated smaller increase in heat-shock resistance was suggested. Survival of males was higher than that of females in all lines when tested with acclimation, but with direct exposure to high temperatures, survival of females was greater than that of males both in selection and control lines but not in the base population. From analysis of reciprocal cross progeny between lines, one selection line was found to possess a dominant autosomal factor that significantly increased resistance of males much more than resistance of females. Also suggestive was recessive traits on the X chromosome in both selection lines that increased thermotolerance. No cytoplasmic effects were found. After accounting for other effects, survival of F1 flies was intermediate, suggesting that additive variation is present for one or more of the autosomes.

Heat-shock resistance in Drosophila populations: analysis of variation in reciprocal cross progeny.

Genetic variation for resistance to high temperature stress was studied in populations of D. melanogaster and D. buzzatii from different geographic regions. Drosophila melanogaster individuals were presented with either a direct short exposure to a high temperature or exposure to high temperature after receiving a pretreatment, which increased resistance. Heat-stress resistance varied among populations, with one much more resistant than all others under both treatments. Another possessed low stress resistance when exposed without the heat pretreatment; but with pretreatment, resistance increased relative to the other populations. Evidence from reciprocal crosses suggests that the X chromosome of the more resistant population carries alleles that greatly increase resistance, and that one or more factors on the autosomes also affect resistance. Non-additive interaction effects among the three less resistant population, were suggestive that all differ for various elements that contribute to stress resistance, and that some clearly change inducible resistance more than basal levels. In D. buzzatii, the two least resistant populations were genetically very similar. Crosses to the more resistant population gave results suggesting that the low resistance to heat is dominant. A small X-chromosome effect that increased resistance, and a dominant enhancer of male resistance also may have contributed to variation in resistance.

Heat-shock tolerance and inbreeding in Drosophila buzzatii.

The effect of inbreeding on survival after a short-term heat shock was tested for two age groups of the cactophilic fruit fly, Drosophila buzzatii, reared under nonstress conditions. Four inbreeding levels (F = 0, F = 0.25, F = 0.375, F = 0.5) were generated by outcrossing or full-sib mating. All flies were conditioned at 36.5 degrees C for 75 min prior to exposure to stress, to activate the synthesis of heat-shock proteins. These proteins are known to protect cells against stress damage. The younger group of flies were exposed to a thermal stress of 40.7 degrees C for 88 min, 103 min, or 118 min and the older flies to the same temperature only for 88 min or 103 min, as the survival of older flies after heat stress was much lower than that of the younger flies. Survival after heat shock declined with increased inbreeding in both age groups. For the younger flies, the slope of the regression line, F, on survival was lower at higher stress levels. For the older flies, inbreeding effects were similar at both stress levels. Mortality without stress also differed significantly among inbreeding groups, mainly because of a large difference between the F = 0.5 group and all others.

Response to environmental change: genetic variation and fitness in Drosophila buzzatii following temperature stress.

Drosophila buzzatii typically may encounter high temperatures in nature, and this species is genetically variable for resistance to stress, both within and among populations. Fitness of survivors to stress, however, was reduced, and observed as a reduction in male fertility and female fecundity. With time following exposure to severe stress, reproductive capacity improved, but lifetime offspring production still was reduced significantly. This effect would greatly reduce a population's recovery from small size, which could occur following exposure to some man-made or environmental extreme. Although the results presented here were obtained for effects of heat stress, such consequences likely apply to a wide range of natural and man-made environmental stresses, including heavy metal toxicity or other pollutants. Low levels of these pollutants may not cause an observable effect on populations, even if some individuals are killed or offspring production is decreased. If genetic variation for resistance is present, higher tolerance may evolve. However, if concentrations are permitted to rise too far, some stress threshold may be reached, as observed for thermal stress, causing mass die-off or sterility and, possibly, local extinction. Understanding the effects of stress is important when preparing programs for the conservation of species. Organisms generally do not become extinct when resources are abundant and the climate benign, but unfortunately, no guarantee can be made that environmental conditions in any locality will remain stable over a long time. Consequently, a high possibility of exposure to an extreme stress in an area would greatly reduce its usefulness as a reserve. Likewise, when choosing organisms for reintroduction, stress resistance of the chosen individuals and high levels of genetic variation within a population would be valuable. The organisms placed there must be able to change. Analysis of stress resistance (at non-lethal levels) among either family groups or for different populations would be very useful when deciding which individuals to reintroduce to an area or place in a reserve. Additionally, analysis may suggest that stress resistance is very low in a population or species. If identified, particular care may be taken to monitor the occurrence of that stress in the environment, and to take action to protect the population from that stress. Preservation of species should be designed for the future, with the goal to preserve, not simply to postpone extinction.

Courtship behavior and control of reproductive isolation in Drosophila mojavensis: genetic analysis of population hybrids.

Drosophila mojavensis from the Sonora region and Baja California show asymmetrical sexual isolation in the laboratory: males from Sonora mate equally frequently with Sonora and Baja females, while the mating success of Baja males with Sonora females is reduced. This failure has been localized to three separate behavioral landmarks occurring during courtship. Genetic analysis was conducted using reciprocal F1 hybrids of Sonora and Baja strains to examine inheritance patterns of the responsible courtship behaviors. Mating success and propensity of F1 males were similar to Sonora males. F1 females mated with males of Sonora and Baja races equally, although mating propensity of F1 females was intermediate between that of Sonora and Baja females. Males of Baja strains presented with F1 females showed a relatively high level of failure at attempted intromission. Genes for mating behaviors are located in the autosomes, but different loci responsible for the sexual isolation appear to act in males and females.

Hypodipsia in geriatric patients.

Hypernatremia in elderly patients is most often due to the patients mental incapacity or physical inability to obtain water despite intact thirst sensation. Hypodipsia leading to hypernatremia is not often considered in alert, elderly subjects since hypodipsia is not a recognized consequence of nonaphasia-producing cerebrovascular accidents. Described herein are six elderly patients who had such cerebrovascular accidents and who had recurrent hospitalizations for dehydration and hypernatremia. Hypernatremia in this group was due to hypodipsia and could only be prevented by prescribing daily fluid intake as a medication order. Hypodipsia should be considered as a cause of hypernatremia in elderly subjects even when they seem fully capable of requesting and obtaining water.

Polyuric prerenal failure.

Prerenal failure is traditionally accompanied by oliguria and represents the normal renal adaptation to retain salt and water and correct the prerenal state. Nonoliguria occurring in the setting of acute renal failure usually represents acute tubular necrosis (ATN) since the kidney has lost its ability to extract salt and water. We report nine cases of patients with acute renal failure occurring in the setting of impaired systemic hemodynamic states and yet who were nonoliguric without strong evidence for ATN. The common defect in these subjects with "polyuric prerenal failure" was a blunted urinary concentrating ability. Polyuria and renal failure occurring despite evidence for impaired systemic hemodynamics may not necessarily be ATN, may still be prerenal, and should be recognizable and promptly reversible if treated appropriately.

Hypomagnesemia. Suppression of secondary hyperparathyroidism in chronic renal failure.

Chronic renal failure is accompanied by secondary hyperparathyroidism. Inhibition of parathyroid hormone secretion has been reported to be induced by hypomagnesemia in conditions other than chronic renal failure, since severe hypomagnesemia is rare in chronic renal failure. In the case reported here, the patient had chronic renal failure and malabsorption-induced hypomagnesemia; she exhibited hypoparathyroidism while hypomagnesemic, and hyperparathyroidism after magnesium was replaced. Hypomagnesemia induced parathyroid hormone suppression in this patient with chronic renal failure, despite the presence of chronic hyperfunctioning parathyroid cells.