The capacity of Drosophila to heat harden associates with low rates of heat-shocked protein synthesis

The thermotolerance of a species or of an ecotype is important for determining its habitat range and vigour, and considerable research has focused on identifying underlying physiological, biochemical and genetic bases of thermotolerance traits. Rates of protein synthesis in tissues when organisms experience a sudden heat stress as occurs on rare hot days may be important to avoid heat-induced paralysis and to survive. While natural variation in Drosophila melanogaster thermotolerance has been associated with heat-shock gene expression, little attention has been given to examining the thermo-protective role of protein synthesis generally. Using two independently derived sets of single-pair mating lines, we characterised variation in rates of protein synthesis in dissected ovarian tissues, both before and after a heat shock applied at different severities in the two sets. In both sets of lines heat-shocked protein synthesis rates were negatively associated with the increase in heat knockdown tolerance after hardening. These associations occurred in a different sex in each set. Variation in rates of Hsp70 synthesis failed to associate with levels of heat tolerance or general protein synthesis. Our results suggest heritable variation in the rate of protein synthesis following heat stress, independently of Hsp70 variation, contributes to heat tolerance variation in this species.     

Dissecting adaptive clinal variation: markers, inversions and size/stress associations in Drosophila melanogaster from a central field population

Many organisms show latitudinal variation for quantitative traits that is assumed to be due to climatic adaptation. These clines provide an opportunity to study the genetics of the adaptive process both at the phenotypic and the underlying molecular levels. Yet researchers rarely try to link variation in quantitative traits to their underlying molecular genetic basis. We describe a novel approach for exploring the genetic basis for clinal variation in size and stress traits in Drosophila melanogaster. We look for associations between genetic markers and traits that exhibit clinal patterns on the east coast of Australia using a single, geographically central population. There are strong associations between markers found within In(3R)Payne and variation in size, suggesting that this inversion explains much of the clinal variation in this trait. We also find that development time is associated with the Adh allozyme locus, cold resistance is negatively associated with the In(3L)Payne inversion and a genetic marker for Hsp70, a heat‐shock protein, is associated with heat resistance. Finally we discuss the importance of inversions in clinal variation for quantitative traits and for identifying quantitative trait loci.     

Temporally regulated expression of a yeast invertase in potato tubers allows dissection of the complex metabolic phenotype obtained following its constitutive expression

The constitutive cytosolic expression of a yeast (Saccharomyces cerevisiae) invertase within potato (Solanum tuberosum) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis. These transgenic lines were further characterised by a massive cycle of sucrose degradation and resynthesis via sucrose-phosphate synthase. We have recently developed a B33 patatin driven alc gene construct allowing tight chemical control of gene expression following supply of acetaldehyde with minimal pleiotropic effects of the inducing agent on metabolism. This construct was used for chemical induction of the yeast invertase gene after 10-weeks growth to dissect the complex metabolic phenotype obtained after constitute expression. Inducible expression led to increased invertase activity within 24 h in well-defined areas within growing tubers. Although the sucrose levels were reduced, there was no effect on the levels of starch whilst levels of many amino acids decreased. Labelling experiments revealed that these lines exhibited increased rates of sucrose cycling, whereas rates of glycolysis and of starch synthesis were not substantially changed. From these results we conclude that sucrose cycling is stimulated in response to a short-term increase in the rate of sucrose mobilisation, providing evidence for a role of sucrose cycling as a buffering capacity that regulates the net rate of sucrose usage. In contrast, the dramatic increase in hexose-phosphate levels and the switch from starch synthesis to respiration seen on the constitutive expression of the invertase was not observed in the inducible lines, suggesting that this is the result of cumulative pleiotropic effects that occurred when the transgene was expressed throughout development.     

Distinct cold tolerance traits independently vary across genotypes in Drosophila melanogaster

Cold tolerance, the ability to cope with low temperature stress, is a critical adaptation in thermally variable environments. An individual's cold tolerance comprises several traits including minimum temperatures for growth and activity, ability to survive severe cold, and ability to resume normal function after cold subsides. Across species, these traits are correlated, suggesting they were shaped by shared evolutionary processes or possibly share physiological mechanisms. However, the extent to which cold tolerance traits and their associated mechanisms covary within populations has not been assessed. We measured five cold tolerance traits—critical thermal minimum, chill coma recovery, short- and long-term cold tolerance, and cold-induced changes in locomotor behavior—along with cold-induced expression of two genes with possible roles in cold tolerance (heat shock protein 70 and frost)—across 12 lines of Drosophila melanogaster derived from a single population. We observed significant genetic variation in all traits, but few were correlated across genotypes, and these correlations were sex-specific. Further, cold-induced gene expression varied by genotype, but there was no evidence supporting our hypothesis that cold-hardy lines would have either higher baseline expression or induction of stress genes. These results suggest cold tolerance traits possess unique mechanisms and have the capacity to evolve independently.     

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.     

Variation in basal heat shock protein 70 is correlated to core temperature in human subjects

Heat shock proteins are highly conserved proteins and play an important chaperone role in aiding the folding of nascent proteins within cells. The heat shock protein response to various stressors, both in vitro and in vivo, is well characterised. However, basal levels of heat shock protein 70 (Hsp70) have not previously been investigated. Monocyte-expressed Hsp70 was determined every 4 h, over a 24 h time period, in 17 healthy male subjects (177 ± 6.4 cm, 75.7 ± 10.9 kg, 19.8 ± 4.3 years) within a temperature and activity controlled environment. Core temperature was measured at 5-min intervals during the 24 h period. Hsp70 showed significant diurnal variation (F = 7.4; p < 0.001), demonstrating peaks at 0900 and 2100 hours, and a nadir at 05.00. Core temperature followed a similar temporal trend (range = 35.96–38.10°C) and was significantly correlated with Hsp70 expression (r _s = 0.44; p < 0.001). These findings suggest a high responsiveness of Hsp70 expression in monocytes to slight variations in core temperature.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Genetic parameters and across‐line SNP associations differ for natural antibody isotypes IgM and IgG in laying hens

In an earlier study, serum levels of natural antibody isotypes IgM‐ and IgG‐binding keyhole limpet hemocyanin were found to be indicative for survival through the laying period of hens and therefore considered as promising traits for future implementation in breeding programs for higher survival of layers. In the present study, we first estimated the genetic parameters for the two isotypes at 20, 40 and 65 weeks of age (IgM20, IgM40 and IgM65; IgG20, IgG40 and IgG65). Pooled genetic parameters were estimated from the total population of 2504 hens from nine purebred layer lines, with line included in the model to account for admixture. Moderate heritabilities (0.14–0.44) indicated that selection for isotype titers is feasible, especially for IgM. Secondly, associations between 1022 SNP markers and the above‐mentioned six immunological traits were estimated in 650 genotyped hens from the nine lines. The association study was performed across lines to detect markers that are closer to the QTL and have the same phase of association in the entire population. Forty‐three significant associations between SNPs and isotype titers were detected. The SNPs of interleukins IL10 and IL19 were associated with both isotypes; SNPs of tripartite motif containing 33 (TRIM33) and IL6 showed significant association with IgG20 and IgM20 respectively; SNPs of heat shock protein 90kDa alpha (cytosolic), class B member 1 (HSP90AB1) were associated with IgG titers at older ages. Some detected SNPs were also reported associated with other immune and behavioral traits.     

Capacity for protein synthesis following heat stimulus of Drosophila associates with heat tolerance but does not underlie the latitudinal tolerance cline

Across populations of Drosophila melanogaster along the Australian eastern coastline latitudinal clines occur in both heat-knockdown tolerance and hardened heat-knockdown tolerance – low latitude tropical populations being more tolerant. A latitudinal cline also occurs for rates of total protein synthesis following a mild heat stress, with tropical populations having higher rates. Since the control of protein synthesis following heat stress is an important component of the cellular heat-shock response, we hypothesised that the higher rates of synthesis that follow a heat stimulus lead to higher knockdown tolerance and underpins the cline. However, levels of heat-stimulated total protein synthesis have been negatively related to heat-hardening capacity, a somewhat conflicting result. Here we examine the relationship between these physiological and adaptive traits in a set of 40 family lines derived from a hybrid laboratory population established by crossing populations from either end of the latitudinal transect. Among these lines high levels of heat-stimulated total protein synthesis were associated with both low basal and low heat-hardened adult knockdown time, confirming the importance of a negative relationship between protein synthesis and thermal tolerance. This result, when considered along with the directions of the latitudinal clines in protein synthesis and tolerance, suggests that variation in rates of heat-stimulated total protein synthesis is not a factor contributing to the latitudinal cline in heat tolerance. Given the robustness of this negative relationship we discuss possible explanations and future experiments to elucidate how the cellular heat stress response might facilitate increased knockdown tolerance.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

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.