Hot and not-so-hot females: reproductive state and thermal preferences of female Arizona Bark Scorpions (Centruroides sculpturatus).

For ectotherms, environmental temperatures influence numerous life history characteristics, and the body temperatures (Tb ) selected by individuals can affect offspring fitness and parental survival. Reproductive trade-offs may therefore ensue for gravid females, because temperatures conducive to embryonic development may compromise females' body condition. We tested whether reproduction influenced thermoregulation in female Arizona Bark Scorpions (Centruroides sculpturatus). We predicted that gravid females select higher Tb and thermoregulate more precisely than nonreproductive females. Gravid C. sculpturatus gain body mass throughout gestation, which exposes larger portions of their pleural membrane, possibly increasing their rates of transcuticular water loss in arid environments. Accordingly, we tested whether gravid C. sculpturatus lose water faster than nonreproductive females. We determined the preferred Tb of female scorpions in a thermal gradient and measured water loss rates using flow-through respirometry. Gravid females preferred significantly higher Tb than nonreproductive females, suggesting that gravid C. sculpturatus alter their thermoregulatory behaviour to promote offspring fitness. However, all scorpions thermoregulated with equal precision, perhaps because arid conditions create selective pressure on all females to thermoregulate effectively. Gravid females lost water faster than nonreproductive animals, indicating that greater exposure of the pleural membrane during gestation enhances the desiccation risk of reproductive females. Our findings suggest that gravid C. sculpturatus experience a trade-off, whereby selection of higher Tb and increased mass during gestation increase females' susceptibility to water loss, and thus their mortality risk. Elucidating the mechanisms that influence thermal preferences may reveal how reproductive trade-offs shape the life history of ectotherms in arid environments.

Gene transcription during exposure to, and recovery from, cold and desiccation stress in Drosophila melanogaster.

We exposed adult male Drosophila melanogaster to cold, desiccation or starvation, and examined expression of several genes during exposure and recovery. Frost was expressed during recovery from cold, and was up-regulated during desiccation. Desiccation and starvation (but not cold) elicited increased expression of the senescence-related gene smp-30. Desat2 decreased during recovery from desiccation, but not in response to starvation or cold. Hsp70 expression increased after 1 h of recovery from cold exposure, but was unchanged in response to desiccation or starvation stress, and Hsp23 levels did not respond to any of the stressors. We conclude that D. melanogaster's responses to cold and desiccation are quite different and that care must be taken to separate exposure and recovery when studying responses to environmental stress.

Effects of starvation and desiccation on energy metabolism in desert and mesic Drosophila.

Energy availability can limit the ability of organisms to survive under stressful conditions. In Drosophila, laboratory experiments have revealed that energy storage patterns differ between populations selected for desiccation and starvation. This suggests that flies may use different sources of energy when exposed to these stresses, but the actual substrates used have not been examined. We measured lipid, carbohydrate, and protein content in 16 Drosophila species from arid and mesic habitats. In five species, we measured the rate at which each substrate was metabolized under starvation or desiccation stress. Rates of lipid and protein metabolism were similar during starvation and desiccation, but carbohydrate metabolism was several-fold higher during desiccation. Thus, total energy consumption was lower in starved flies than desiccated ones. Cactophilic Drosophila did not have greater initial amounts of reserves than mesic species, but may have lower metabolic rates that contribute to stress resistance.

Evolution of water balance in the genus Drosophila.

Fruit flies of the genus Drosophila have independently invaded deserts around the world on numerous occasions. To understand the physiological mechanisms allowing these small organisms to survive and thrive in arid environments, we performed a phylogenetic analysis of water balance in Drosophila species from different habitats. Desert (cactophilic) species were more resistant to desiccation than mesic ones. This resistance could be accomplished in three ways: by increasing the amount of water in the body, by reducing rates of water loss or by tolerating the loss of a greater percentage of body water (dehydration tolerance). Cactophilic Drosophila lost water less rapidly and appeared to be more tolerant of low water content, although males actually contained less water than their mesic congeners. However, when the phylogenetic relationships between the species were taken into account, greater dehydration tolerance was not correlated with increased desiccation resistance. Therefore, only one of the three expected adaptive mechanisms, lower rates of water loss, has actually evolved in desert Drosophila, and the other apparently adaptive difference between arid and mesic species (increased dehydration tolerance) instead reflects phylogenetic history.

Effects of age on water balance in Drosophila species.

Age-related declines in physiological performance have been documented in a wide variety of organisms. However, it is unknown whether related species age in a similar manner or whether physiological differences associated with aging differ widely among species. Previous work has shown that the desiccation resistance of Drosophila melanogaster decreases rapidly with age. Other Drosophila species may have delayed reproductive maturity or may inhabit arid habitats, so that age-related changes in water balance may be a significant physiological constraint on their ecological success. We may, therefore, predict that physiological systems involved in water balance will deteriorate with age at a reduced rate in these species. We quantified several components of water budgets for 14 species of Drosophila, including both desert and mesic representatives differing in their age of maturity. Desiccation resistance decreased with age in all but one species studied, primarily because of increased rates of water loss. However, there was no significant relationship between the rate of aging, as indicated by the rate of increase in water-loss rates as flies aged, and either habitat or age of maturity.

Chemical and physical analyses of wax ester properties.

Wax esters are major constituents of the surface lipids in many terrestrial arthropods, but their study is complicated by their diversity. We developed a procedure for quantifying isomers in mixtures of straight-chain saturated and unsaturated wax esters having the same molecular weights, using single-ion monitoring of the total ion current data from gas chromatography-mass spectrometry. We examined the biological consequences of structural differences by measuring the melting temperatures, T(m), of >60 synthetic wax esters, containing 26-48 carbon atoms. Compounds containing saturated alcohol and acid moieties melted at 38-73 degrees C. The main factor affecting T(m) was the total chain length of the wax ester, but the placement of the ester bond also affected T(m). Insertion of a double bond into either the alcohol or acid moiety decreased T(m) by approximately 30 degrees C. Simple mixtures of wax esters with n-alkanes melted several degrees C lower than predicted from the melting points of the component lipids. Our results indicate that the wax esters of primary alcohols that are most typically found on the cuticle of terrestrial arthropods occur in a solid state under physiological conditions, thereby conferring greater waterproofing. Wax esters of secondary alcohols, which occur on melanopline grasshoppers, melted >60 degrees C below primary esters of the same molecular weight and reduced T(m) of the total surface lipids to environmental values.

Postponed aging and desiccation resistance in Drosophila melanogaster.

Studies with the fruit fly, Drosophila melanogaster, have repeatedly shown that selection for postponed reproduction leads to increases in mean life span and increased stress resistance; including increased resistance to desiccation, starvation and ethanol vapors. We show that desiccation resistance declines with age in both short- and long-lived flies suggesting that desiccation resistance may serve as a useful biomarker for aging-related declines in physiological performance. We examined the physical basis of desiccation resistance in five replicate populations selected for postponed reproduction and five replicate control populations. The variables examined were water content, rates of water loss during desiccation, and water content at time of death due to desiccation. In the absence of desiccation stress, both the flies exhibiting postponed senescence and their controls maintained constant water content throughout their lifetimes. In the presence of desiccation stress, the short-lived flies showed significantly higher rates of water loss at all ages than did the long-lived flies. Flies from the two treatments did not differ in water content at death. Our results indicate that water loss rates are the major determinant of desiccation resistance. Water loss rates are under genetic control and covary with age in populations with genetically-determined postponed senescence.

Laboratory selection for the comparative physiologist.

An increasingly popular experimental approach in comparative physiology is to study the evolution of physiological traits in the laboratory, using microbial, invertebrate and vertebrate models. Because selective conditions are well-defined, selected populations can be replicated and unselected control populations are available for direct comparison, strong conclusions regarding the adaptive value of an evolved response can be drawn. These studies have shown that physiological systems evolve rapidly in the laboratory, but not always as one would expect from comparative studies of different species. Laboratory environments are often not as simple as one thinks, so that the evolution of behavioral differences or selection acting on different life stages can lead to unanticipated results. In some cases, unexpected responses to laboratory selection may suggest new insights into physiological mechanisms, which might not be available using other experimental approaches. I outline here recent results (including success stories and caveats for the unwary investigator) and potential directions for selection experiments in comparative physiology.

Osmoregulation in Drosophila melanogaster selected for urea tolerance.

Animals may adapt to hyperosmolar environments by either osmoregulating or osmoconforming. Osmoconforming animals generally accumulate organic osmolytes including sugars, amino acids or, in a few cases, urea. In the latter case, they also accumulate 'urea-counteracting' solutes to mitigate the toxic effects of urea. We examined the osmoregulatory adaptation of Drosophila melanogaster larvae selected to live in 300 mmol l(-)(1) urea. Larvae are strong osmoregulators in environments with high NaCl or sucrose levels, but have increased hemolymph osmolarity on urea food. The increase in osmolarity on urea food is smaller in the selected larvae relative to unselected control larvae, and their respective hemolymph urea concentrations can account for the observed increases in total osmolarity. No other hemolymph components appear to act as urea-counteractants. Urea is calculated to be in equilibrium across body compartments in both selected and control larvae, indicating that the selected larvae are not sequestering it to lower their hemolymph osmolarity. The major physiological adaptation to urea does not appear to involve increased tolerance or improved osmoregulation per se, but rather mechanisms (e.g. metabolism, decreased uptake or increased excretion) that reduce overall urea levels and the consequent toxicity.

The effect of urea exposure on isoaspartyl content and protein L-isoaspartate methyltransferase activity in Drosophila melanogaster.

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (beta-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein L-isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.

Effects of temperature on cuticular lipids and water balance in a desert Drosophila: is thermal acclimation beneficial?

The desert fruit fly Drosophila mojavensis experiences environmental conditions of high temperature and low humidity. To understand the physiological mechanisms allowing these small insects to survive in such stressful conditions, we studied the effects of thermal acclimation on cuticular lipids and rates of water loss of adult D. mojavensis. Mean hydrocarbon chain length increased at higher temperatures, but cuticular lipid melting temperature (Tm) did not. Lipid quantity doubled in the first 14 days of adult life, but was unaffected by acclimation temperature. Despite these changes in cuticular properties, organismal rates of water loss were unaffected by either acclimation temperature or age. Owing to the smaller body size of warm-acclimated flies, D. mojavensis reared for 14 days at 33 degrees C lost water more rapidly on a mass-specific basis than flies acclimated to 25 degrees C or 17 degrees C. Thus, apparently adaptive changes in cuticular lipids do not necessarily result in reduced rates of water loss. Avoidance of high temperatures and desiccating conditions is more likely to contribute to survival in nature than changes in water balance mediated by surface lipids.

A genetic polymorphism maintained by natural selection in a temporally varying environment.

Environments that are crowded with larvae of the fruit fly, Drosophila melanogaster, exhibit a temporal deterioration in quality as waste products accumulate and food is depleted. We show that natural selection in these environments can maintain a genetic polymorphism with one group of genotypes specializing on the early part of the environment and a second group specializing on the late part. These specializations involve trade-offs in fitness components. The early types emerge first from crowded cultures and have high larval feeding rates, which are positively correlated with competitive ability but exhibit lower absolute viability than the late phenotype, especially in food contaminated with the nitrogenous waste product, ammonia. The late emerging types have reduced feeding rates but higher absolute survival under conditions of severe crowding and high levels of ammonia. Organisms that experience temporal variation within a single generation are not uncommon, and this model system provides some of the first insights into the evolutionary forces at work in these environments.

Physiological mechanisms of evolved desiccation resistance in Drosophila melanogaster.

We investigated physiological characters associated with water balance in laboratory populations of Drosophila melanogaster selected for resistance to desiccating conditions for over 100 generations. Five replicate, outbred, desiccation-selected (D) populations were compared with their control (C) populations. Water loss rates of female D flies were approximately 40% lower than those of C females. Although excretory water loss was reduced in desiccation-selected flies, it comprised less than 10% of total water loss, indicating that the D populations have evolved reduced cuticular and/or respiratory water loss rates. Total surface lipid amounts did not differ between the C and D flies. Cuticular hydrocarbons from D flies were longer than those from C flies and melted at slightly higher temperatures, possibly contributing to reduced water loss rates. Desiccation-selected flies contained approximately 30% more bulk water than controls, as well as more glycogen. However, total metabolic water stores did not differ between the stocks owing to higher lipid levels in the C populations. The ability to tolerate water loss, as measured by water content at the time of death, did not differ between D and C flies. Thus, evolution of increased desiccation resistance has occurred by multiple physiological mechanisms, but some potential adaptive differences have not evolved.

Functional conflicts between feeding and gas exchange in suspension-feeding tadpoles, Xenopus laevis.

Air-breathing tadpoles of Xenopus laevis (Amphibia: Anura) use buccopharyngeal surfaces for both gas exchange and capture of food particles in the water. In dense food suspensions, tadpoles decrease ventilation of the buccopharynx and increase air breathing. The lung ventilatory frequency is elevated even though the rate of oxygen consumption is at or below resting levels, suggesting that the lung hyperventilation reflects compensation for decreased buccopharyngeal respiration rather than an increased metabolic requirement. If tadpoles in hypoxic water are prevented from breathing air, they increase buccopharyngeal respiration at the expense of feeding. Aerial respiration evidently permits the buccopharyngeal surfaces to be used primarily for food entrapment.

Resistance of the shell membrane and mineral layer to diffusion of oxygen and water in flexible-shelled eggs of the snapping turtle (Chelydra serpentina).

At oviposition, flexible eggshells of many turtles have an outer mineral layer and an inner membrane layer of approximately equal thickness. We measured conductances of both layers to H2O and O2 at various levels of eggshell hydration. Both the mineral layer of the eggshell and the shell membrane offer significant resistance to diffusion of water vapor and oxygen in eggshells of the snapping turtle, Chelydra serpentina. Conductance to water vapor increases in both the membrane and mineral layer with increasing hydration of the eggshell, but conductance to oxygen decreases under similar conditions. Removal of the mineral layer increases conductance to oxygen in moist and dry eggshells, but decreases conductance at intermediate levels of dehydration. Removal of the mineral layer consistently increases conductance to water vapor. The eggshell membrane accounts for 24-76% of overall resistance to diffusion of water vapor. These results suggest that bulk flow of H2O or physical changes in the shell may interact with diffusion to limit gas exchange through the turtle eggshell.