Robert A. Krebs

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.

Costs and benefits of activation of the heat-shock response in Drosophila melanogaster

1. The costs of conditioning adult Drosophila melanogaster with a mild thermal stress that activates the genes for heat-shock proteins were examined by comparing the number of offspring produced by females maintained continuously at 25 o C with females exposed to a non-lethal stress, 36 o C for 75 min, once, twice or three times. The comparison was done under two nutritional treatments, with or without yeast added to the medium. 2. Benefits of conditioning adult D. melanogaster to thermal stress were examined by comparing survival after a severe stress (39 o C for 100 min) among flies that were not conditioned with those conditioned once, twice or three times by exposure to 36 o C for 75 min

NATURAL VARIATION IN THE EXPRESSION OF THE HEAT-SHOCK PROTEIN HSP70 IN A POPULATION OF DROSOPHILA MELANOGASTER AND ITS CORRELATION WITH TOLERANCE OF ECOLOGICALLY RELEVANT THERMAL STRESS

Although Hsp70, the principal inducible heat‐shock protein of Drosophila melanogaster, has received intense scrutiny in laboratory strains, its variation within natural populations and the consequences of such variation for thermotolerance are unknown. We have characterized variation in first‐instar larvae of 20 isofemale lines isolated from a single natural population of D. melanogaster, in which larvae are prone to thermal stress in nature. Hsp70 expression varied more than twofold among lines after induction by exposure to 36°C for one hour, with an estimated proportion of the variation due to genetic differences of 0.24 ± 0.08. Thermotolerance with and without a Hsp70‐inducing pretreatment, survival at 25°C, and developmental time also varied significantly. As expected, expression of Hsp70 correlated positively with larval thermotolerance. By contrast, lines in which larval survival was high in the absence of heat stress showed lower than average Hsp70 expression and lower than average inducible thermotolerance. This conditional performance suggests an evolutionary trade‐off between thermotolerance and the ability to produce higher concentrations of Hsp70, and survival in a benign environment.

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.

Effects of exposure to short‐term heat stress on fitness components in Drosophila melanogaster

Effects of thermal stress on survival and reproductive success in ten recently collected isofemale lines of Drosophila melanogaster were compared for flies treated as follows: always held at 25° C, placed in an incubator set at 37° C for 120 min, or exposed to 40° C in an incubator for 90 min, with or without previous exposure to 37° C. Short‐term exposure to the higher temperature greatly reduced adult survival, the mating frequency of males and females, and female fecundity, which was measured as offspring produced over ten days. Male fertility, measured as the progeny produced by a female mated once, differed little among treatments. Previous exposure to a high, but non‐lethal, temperature before exposure to the higher one, improved survival of males and females, and improved offspring production of females. Genetic variation was present among lines for offspring production, but genetic variation for survival was not significant, and genotype by environment interactions for fitness components of females were small. These results indicated low genetic variation in thermal resistance in the studied population, such that a threshold for temperature stress probably exists, above which local extinction is more likely than the evolution of resistance.

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.

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.

Genetic variation and plasticity of thorax length and wing length in Drosophila aldrichi and D. buzzatii

Reaction norms across three temperatures of development were measured for thorax length, wing length and wing length/thorax length ratio for ten isofemale lines from each of two populations of Drosophila aldrichi and D. buzzatii. Means for thorax and wing length in both species were larger at 24 °C than at either 18 °C or 31 °C, with the reduction in size at 18 °C most likely due to a nutritional constraint. Although females were larger than males, the sexes were not different for wing length/thorax length ratio. The plasticity of the traits differed between species and between populations of each species, with genetic variation in plasticity similar for the two species from one locality, but much higher for D. aldrichi from the other. Estimates of heritabilities for D. aldrichi generally were higher at 18 °C and 24 °C than at 31 °C, but for D. buzzatii they were highest at 31 °C, although heritabilities were not significantly different between species at any temperature. Additive genetic variances for D. aldrichi showed trends similar to that for heritability, being highest at 18 °C and decreasing as temperature increased. For D. buzzatii, however, additive genetic variances were lowest at 24 °C. These results are suggestive that genetic variation for body size characters is increased in more stressful environments. Thorax and wing lengths showed significant genetic correlations that were not different between the species, but the genetic correlations between each of these traits and their ratio were significantly different. For D. aldrichi, genetic variation in the wing length/thorax length ratio was due primarily to variation in thorax length, while for D. buzzatii, it was due primarily to variation in wing length. The wing length/thorax length ratio, which is the inverse of wing loading, decreased linearly as temperature increased, and it is suggested that this ratio may be of greater adaptive significance than either of its components.

Heritability of Expression of the 70kd Heat-shock Protein in Drosophila melanogaster and its Relevance to the Evolution of Thermotolerance

The principle inducible heat-shock protein of Drosophila melanogaster, Hsp70, contributes to thermotolerance throughout the entire life cycle of the species but may also reduce fitness in some life stages. In principle, selection might maximize the benefits of Hsp70 expression relative to its costs by adjusting the magnitude of Hsp70 expression for each life-cycle stage independently. Therefore we examined whether the magnitude of Hsp70 expression varied during the life cycle and the relationship of this variation to several life-history traits. For 28 isofemale lines derived from a single natural population, estimates of heritable variation in Hsp70 expression ranged between 0.25 and 0.49, and the association among variation in first- and third-instar larvae and in adults correlated highly. Thus, Hsp70 expression is genetically coupled at these developmental stages. A line engineered with extra copies of the hsp70 gene produced more Hsp70 and survived heat shock much better than did a control strain. Among natural lines, Hsp70 expression was only weakly related to tolerance of heat shock and to larva-to-adult survival and developmental time at permissive temperatures. Additionally, lines with high adult survival developed slowly as larvae, which is a possible trade-off. These and other findings suggest that trade-offs may maintain quantitative variation both in heat-shock protein expression and in life-history traits that associate with thermotolerance.

SELECTION FOR HEAT-SHOCK RESISTANCE IN LARVAL AND IN ADULT DROSOPHILA BUZZATII : COMPARING DIRECT AND INDIRECT RESPONSES

Direct and correlated responses in selection for heat‐shock resistance in adult and in larval Drosophila buzzatii were studied. Two lines were artificially selected for higher survival to heat stress as adults, and two other lines were reared under a fluctuating thermal environment as larvae, 35°C for 6 h and 25°C for 18 h, to “naturally” select for higher resistance as larvae. The latter two lines were duplicated after nine generations to yield additional lines to be “naturally” selected as larvae at a higher temperature, 38.2°C for 6 h. Control lines were maintained separately for the adult and larval selection lines. A significant direct response to selection was found for the adult selection lines. However, larvae of these adult selection lines were no more heat resistant than were larvae of the control lines. One of the two larval selection lines increased significantly in heat resistance as larvae. However, adult heat resistance was similar for lines selected as larvae and the corresponding control lines maintained at 25°C. Changes in developmental time accompanied changes in survival after stress in both sets of lines selected for increased heat resistance.