Within-population plastic responses to combined thermal-nutritional stress differ from those in response to single stressors, and are genetically independent across traits in both males and females.

Phenotypic plasticity helps animals to buffer the effects of increasing thermal and nutritional stress created by climate change. Plastic responses to single and combined stressors can vary among genetically diverged populations. However, less is known about how plasticity in response to combined stress varies among individuals within a population or whether such variation changes across life-history traits. This is important because individual variation within populations shapes population-level responses to environmental change. Here, we used isogenic lines of Drosophila melanogaster to assess the plasticity of egg-to-adult viability and sex-specific body size for combinations of 2 temperatures (25 °C or 28 °C) and 3 diets (standard diet, low caloric diet, or low protein:carbohydrate ratio diet). Our results reveal substantial within-population genetic variation in plasticity for egg-to-adult viability and wing size in response to combined thermal-nutritional stress. This genetic variation in plasticity was a result of cross-environment genetic correlations that were often < 1 for both traits, as well as changes in the expression of genetic variation across environments for egg-to-adult viability. Cross-sex genetic correlations for body size were weaker when the sexes were reared in different conditions, suggesting that the genetic basis of traits may change with the environment. Furthermore, our results suggest that plasticity in egg-to-adult viability is genetically independent from plasticity in body size. Importantly, plasticity in response to diet and temperature individually differed from plastic shifts in response to diet and temperature in combination. By quantifying plasticity and the expression of genetic variance in response to combined stress across traits, our study reveals the complexity of animal responses to environmental change, and the need for a more nuanced understanding of the potential for populations to adapt to ongoing climate change.

Short-term fasting of a single amino acid extends lifespan.

Diet and health are strongly linked, though the strict changes in diet required to improve health outcomes are usually difficult to sustain. We sought to understand whether short-term bouts of amino acid-specific modifications to the diet of Drosophila melanogaster could mimic the lifespan and stress resistance benefits of dietary restriction, without the requirement for drastic reductions in food intake. We found that flies that were transiently fed diets lacking the essential amino acid isoleucine, but otherwise nutritionally complete, exhibited enhanced nicotine tolerance, indicating elevated detoxification capacity. The protection from isoleucine deprivation increased with the duration of exposure, up to a maximum at 7-day isoleucine deprivation for flies 2, 3, or 4 weeks of age, and a 5-day deprivation when flies were 5 weeks of age. Because of these beneficial effects on toxin resistance, we intermittently deprived flies of isoleucine during the first 6 weeks of adulthood and monitored the effect on lifespan. Lifespan was significantly extended when flies experienced short-term isoleucine deprivation at 3 and 5 weeks of age, regardless of whether they were also deprived at 1 week. These results indicate that short-term bouts of isoleucine deprivation can extend lifespan and highlight its cumulative and time-dependent benefits. Interestingly, we found that isoleucine-deprived flies lost their protection against nicotine within 3 days of returning to fully fed conditions. Therefore, the mechanisms underlying lifespan extension may involve transient damage clearance during the bouts of isoleucine deprivation rather than sustained enhanced detoxification capacity. These data highlight a new time-restricted, nutritionally precise method to extend life in Drosophila melanogaster and point to a more manageable dietary method to combat ageing.

Timing Drosophila development through steroid hormone action.

Specifically timed pulses of the moulting hormone ecdysone are necessary for developmental progression in insects, guiding development through important milestones such as larval moults, pupation and metamorphosis. It also coordinates the acquisition of cell identities, known as cell patterning, and growth in a tissue-specific manner. In the absence of ecdysone, the ecdysone receptor heterodimer Ecdysone Receptor and Ultraspiracle represses expression of target primary response genes, which become de-repressed as the ecdysone titre rises. However, ecdysone signalling elicits both repressive and activating responses in a temporal and tissue-specific manner. To understand how ecdysone achieves such specificity, this review explores the layers of gene regulation involved in stage-appropriate ecdysone responses in Drosophila fruit flies.

Temperature and nutrition do not interact to shape the evolution of metabolic rate.

Metabolic cold adaptation, or Krogh's rule, is the controversial hypothesis that predicts a monotonically negative relationship between metabolic rate and environmental temperature for ectotherms living along thermal clines measured at a common temperature. Macrophysiological patterns consistent with Krogh's rule are not always evident in nature, and experimentally evolved responses to temperature have failed to replicate such patterns. Hence, temperature may not be the sole driver of observed variation in metabolic rate. We tested the hypothesis that temperature, as a driver of energy demand, interacts with nutrition, a driver of energy supply, to shape the evolution of metabolic rate to produce a pattern resembling Krogh's rule. To do this, we evolved replicate lines of Drosophila melanogaster at 18, 25 or 28°C on control, low-calorie or low-protein diets. Contrary to our prediction, we observed no effect of nutrition, alone or interacting with temperature, on adult female and male metabolic rates. Moreover, support for Krogh's rule was only in females at lower temperatures. We, therefore, hypothesize that observed variation in metabolic rate along environmental clines arises from the metabolic consequences of environment-specific life-history optimization, rather than because of the direct effect of temperature on metabolic rate. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing.

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

Biosynthetic constraints on amino acid synthesis at the base of the food chain may determine their use in higher-order consumer genomes.

Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited, and for Drosophila melanogaster this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumer's exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. Here, we update the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, we found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, we also found that exome matched AA profiles are generally conserved across taxa and that the composition of these profiles might be explained by energetic and elemental limitations on microbial AA synthesis. Thus, it appears that ecological constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that this constrains biomass composition.

Drosophila melanogaster females prioritise dietary sterols for producing viable eggs.

Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan and reduce reproduction across a broad range of taxa. Our recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR, while also sustaining high egg production. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mother's demand for sterols, thus supporting longer lifespan. It is also possible that mothers live longer and lay more eggs on high sterol diets because the diet triggers enhanced somatic maintenance and promotes egg production, but at the cost of diminished egg quality. To test this, we measured the viability of eggs and development of offspring from mothers fed either cholesterol-sufficient or cholesterol-limiting diets. We found that even when the mother's diet was completely devoid of cholesterol, viable egg production persisted for ∼10 days. Furthermore, we show that sterol-supplemented flies with long lives lay eggs that have high viability and the same developmental potential as those laid by shorter lived mothers on sterol limiting diets. These findings suggest that offspring viability is not a hidden cost of lifespan extension seen in response to dietary sterol supplementation.

Rethinking the ecdysteroid source during Drosophila pupal-adult development.

Ecdysteroids, typified by 20-hydroxyecdysone (20E), are essential hormones for the development, reproduction and physiology of insects and other arthropods. For over half a century, the vinegar fly Drosophila melanogaster (Ephydroidea: Diptera) has been used as a model of ecdysteroid biology. Many aspects of the biosynthesis and regulation of ecdysteroids in this species are understood at the molecular level, particularly with respect to their secretion from the prothoracic gland (PG) cells of the ring gland, widely considered the dominant biosynthetic tissue during development. Discrete pulses of 20E orchestrate transitions during the D. melanogaster life cycle, the sources of which are generally well understood, apart from the large 20E pulse at the onset of pharate adult development, which has received little recent attention. As the source of this pharate adult pulse (PAP) is a curious blind spot in Drosophila endocrinology, we evaluate published biochemical and genetic data as they pertain to three hypotheses for the source of PAP 20E: the PG; an alternative biosynthetic tissue; or the recycling of stored 20E. Based on multiple lines of evidence, we contend the PAP cannot be derived from biosynthesis, with other data consistent with D. melanogaster able to recycle ecdysteroids before and during metamorphosis. Published data also suggest the PAP is conserved across Diptera, with evidence for pupal-adult ecdysteroid recycling occurring in other cyclorrhaphan flies. Further experimental work is required to test the ecdysteroid recycling hypothesis, which would establish fundamental knowledge of the function, regulation, and evolution of metamorphic hormones in dipterans and other insects.

Dietary restriction and lifespan: adaptive reallocation or somatic sacrifice?

Reducing overall food intake, or lowering the proportion of protein relative to other macronutrients, can extend the lifespan of diverse organisms. A number of mechanistic theories have been developed to explain this phenomenon, mostly assuming that the molecules connecting diet to lifespan are evolutionarily conserved. A recent study using Drosophila melanogaster females has pinpointed a single essential micronutrient that can explain how lifespan is changed by dietary restriction. Here, we propose a likely mechanism for this observation, which involves a trade-off between lifespan and reproduction, but in a manner that is conditional on the dietary supply of an essential micronutrient - a sterol. Importantly, these observations argue against previous evolutionary theories that rely on constitutive resource reallocation or damage directly inflicted by reproduction. Instead, they are compatible with a model in which the inverse relationship between lifespan and food level is caused by the consumer suffering from varying degrees of malnutrition when maintained on lab food. The data also indicate that animals on different lab foods may suffer from different nutritional imbalances and that the mechanisms by which dietary restriction benefits the lifespan of different species may vary. This means that translating the mechanistic findings from lab animals to humans will not be simple and should be interpreted in light of the range of challenges that have shaped each organism's lifespan in the wild and the composition of the natural diets upon which they would feed.

Within-population variation in body size plasticity in response to combined nutritional and thermal stress is partially independent from variation in development time.

Ongoing climate change has forced animals to face changing thermal and nutritional environments. Animals can adjust to such combinations of stressors via plasticity. Body size is a key trait influencing organismal fitness, and plasticity in this trait in response to nutritional and thermal conditions varies among genetically diverse, locally adapted populations. The standing genetic variation within a population can also influence the extent of body size plasticity. We generated near-isogenic lines from a newly collected population of Drosophila melanogaster at the mid-point of east coast Australia and assayed body size for all lines in combinations of thermal and nutritional stress. We found that isogenic lines showed distinct underlying patterns of body size plasticity in response to temperature and nutrition that were often different from the overall population response. We then tested whether plasticity in development time could explain, and therefore regulate, variation in body size to these combinations of environmental conditions. We selected five genotypes that showed the greatest variation in response to combined thermal and nutritional stress and assessed the correlation between response of developmental time and body size. While we found significant genetic variation in development time plasticity, it was a poor predictor of body size among genotypes. Our results therefore suggest that multiple developmental pathways could generate genetic variation in body size plasticity. Our study emphasizes the need to better understand genetic variation in plasticity within a population, which will help determine the potential for populations to adapt to ongoing environmental change.

Evaluating old truths: Final adult size in holometabolous insects is set by the end of larval development.

For centuries, it has been understood that the final size of adult holometabolous insects is determined by the end of the larval stage, and that once they transform to adults, holometabolous insects do not grow. Despite this, no previous study has directly tested these "old truths" across holometabolous insects. Here, we demonstrate that final adult size is set at the end of the last larval stage in species representing each of the four orders of holometabolous insects: the fruit fly Drosophila melanogaster (Diptera), the tobacco hornworm Manduca sexta (Lepidoptera), the dung beetle Onthophagus taurus (Coleoptera), and the Florida carpenter ant Camponotus floridanus (Hymenoptera). Furthermore, in both D. melanogaster and C. floridanus, we show that the size of adult individuals fluctuates but does not significantly change. Therefore, our study finally confirms these two basic assumptions in the biology of insects, which have for centuries served as the foundation for studies of insect growth, size, and allometry.

Restricting a single amino acid cross-protects Drosophila melanogaster from nicotine poisoning through mTORC1 and GCN2 signalling.

Dietary interventions that restrict protein intake have repeatedly been shown to offer beneficial health outcomes to the consumer. Benefits such as increased stress tolerance can be observed when individual amino acids are restricted, thus mimicking dietary protein restriction. Here, we sought to further understand the relationship between dietary amino acids and stress tolerance using Drosophila melanogaster. Using a chemically defined medium for Drosophila, we found that transiently restricting adult flies of a single essential amino acid generally protects against a lethal dose of the naturally occurring insecticide, nicotine. This protection varied with the identity of the focal amino acid and depended on the duration and intensity of its restriction. To understand the molecular basis of these effects, we modified the signalling of two cellular sensors of amino acids, GCN2 and mTORC1, in combination with amino acid restriction. We found that GCN2 was necessary for diets to protect against nicotine, whereas the suppression of mTORC1 was sufficient to induce nicotine resistance. This finding implies that amino acid restriction acts via amino acid signalling to cross-protect against seemingly unrelated stressors. Altogether, our study offers new insights into the physiological responses to restriction of individual amino acids that confer stress tolerance.

Maintaining robust size across environmental conditions through plastic brain growth dynamics.

Organ growth is tightly regulated across environmental conditions to generate an appropriate final size. While the size of some organs is free to vary, others need to maintain constant size to function properly. This poses a unique problem: how is robust final size achieved when environmental conditions alter key processes that regulate organ size throughout the body, such as growth rate and growth duration? While we know that brain growth is 'spared' from the effects of the environment from humans to fruit flies, we do not understand how this process alters growth dynamics across brain compartments. Here, we explore how this robustness in brain size is achieved by examining differences in growth patterns between the larval body, the brain and a brain compartment-the mushroom bodies-in Drosophila melanogaster across both thermal and nutritional conditions. We identify key differences in patterns of growth between the whole brain and mushroom bodies that are likely to underlie robustness of final organ shape. Further, we show that these differences produce distinct brain shapes across environments.

Target of Rapamycin Drives Unequal Responses to Essential Amino Acid Depletion for Egg Laying in Drosophila Melanogaster.

Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, we fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, we tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, we found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. Our results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal's true nutritional status.

When does diet matter? The roles of larval and adult nutrition in regulating adult size traits in Drosophila melanogaster.

Adult body size is determined by the quality and quantity of nutrients available to animals. In insects, nutrition affects adult size primarily during the nymphal or larval stages. However, measures of adult size like body weight are likely to also change with adult nutrition. In this study, we sought to explore the roles of nutrition throughout the life cycle on adult body weight and the size of two appendages, the wing and the femur, in the fruit fly Drosophila melanogaster. We manipulated nutrition in two ways: by varying the protein to carbohydrate content of the diet, called macronutrient restriction, and by changing the caloric density of the diet, termed caloric restriction. We employed a fully factorial design to manipulate both the larval and adult diets for both diet types. We found that manipulating the larval diet had greater impacts on all measures of adult size. Further, macronutrient restriction was more detrimental to adult size than caloric restriction. For adult body weight, a rich adult diet mitigated the negative effects of poor larval nutrition for both types of diets. In contrast, small wing and femur size caused by poor larval diet could not be increased with the adult diet. Taken together, these results suggest that appendage size is fixed by the larval diet, while those related to body composition remain sensitive to adult diet. Further, our studies provide a foundation for understanding how the nutritional environment of juveniles affects how adults respond to diet.

Amino acid quality modifies the quantitative availability of protein for reproduction in Drosophila melanogaster.

Diet composition, especially the relative abundance of key macronutrients, is well known to affect animal wellbeing by changing reproductive output, metabolism and length of life. However, less attention has been paid to the ways the quality of these nutrients modify these macronutrient interactions. Nutritional Geometry can be used to model the effects of multiple dietary components on life-history traits and to compare these responses when diet quality is varied. Previous studies have shown that dietary protein quality can be increased for egg production in Drosophila melanogaster by matching the dietary amino acid proportions to the balance of amino acids used by the sum of proteins in the fly's in silico translated exome. Here, we show that dietary protein quality dramatically alters the effect of protein quantity on female reproduction across a broad range of diets varying in both protein and carbohydrate concentrations. These data show that when sources of ingredients vary, their relative value to the consumer can vastly differ and yield very different physiological outcomes. Such variations could be particularly important for meta analyses that look to draw generalisable conclusions from diverse studies.

The proximate sources of genetic variation in body size plasticity: The relative contributions of feeding behaviour and development in Drosophila melanogaster.

Body size is a key life-history trait that influences many aspects of an animal's biology and is shaped by a variety of factors, both genetic and environmental. While we know that locally-adapted populations differ in the extent to which body size responds plastically to environmental conditions like diet, we have a limited understanding of what causes these differences. We hypothesized that populations could differ in the way body size responds to nutrition either by modulating growth rate, development time, feeding rate, or a combination of the above. Using three locally-adapted populations of Drosophila melanogaster from along the east coast of Australia, we investigated body size plasticity across five different diets. We then assessed how these populations differed in feeding behaviour and developmental timing on each of the diets. We observed population-specific plastic responses to nutrition for body size and feeding rate, but not development time. However, differences in feeding rate did not fully explain the differences in the way body size responded to diet. Thus, we conclude that body size variation in locally-adapted populations is shaped by a combination of growth rate and feeding behaviour. This paves the way for further studies that explore how differences in the regulation of the genetic pathways that control feeding behaviour and growth rate contribute to population-specific responses of body size to diet.

Ecdysone Quantification from Whole Body Samples of Drosophila melanogaster Larvae.

Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass in insects, and the duration of this growth period is driven by a sequence of small ecdysone pulses that ultimately induce metamorphosis. Historically the biologically active form of ecdysone, 20-hydroxyecdysone (20E), was quantified using radio-immunoassays, bioassays, or chromatography assays. However, these assays are methodologically complicated and often time consuming. Furthermore, collecting samples for precise measurements of ecdysone concentrations using these assays is limited in small insects like Drosophila melanogaster. Here, we describe an accurate and sensitive method to collect carefully-staged third instar larvae suitable for preparing samples for ecdysone quantification using a commercially-available 20E enzyme immunoassay (EIA). Because we resynchronize larval development at the molt to the final instar, collect large samples, and weigh each sample, we are able to detect a small ecdysone peak early in the final instar known as the critical weight ecdysone peak. This method detects peaks as low as 6 pg 20E/mg larval sample, allowing us to quantify other small ecdysone peaks in flies - the necessary prerequisite for eventually determining their regulation and function.

Regulation of ecdysone production in Drosophila by neuropeptides and peptide hormones.

In both mammals and insects, steroid hormones play a major role in directing the animal's progression through developmental stages. To maximize fitness outcomes, steroid hormone production is regulated by the environmental conditions experienced by the animal. In insects, the steroid hormone ecdysone mediates transitions between developmental stages and is regulated in response to environmental factors such as nutrition. These environmental signals are communicated to the ecdysone-producing gland via the action of neuropeptide and peptide hormone signalling pathways. While some of these pathways have been well characterized, there is evidence to suggest more signalling pathways than has previously been thought function to control ecdysone production, potentially in response to a greater range of environmental conditions. Here, we review the neuropeptide and peptide hormone signalling pathways known to regulate the production of ecdysone in the model genetic insect Drosophila melanogaster, as well as what is known regarding the environmental signals that trigger these pathways. Areas for future research are highlighted that can further contribute to our overall understanding of the complex orchestration of environmental, physiological and developmental cues that together produce a functioning adult organism.

A dietary sterol trade-off determines lifespan responses to dietary restriction in Drosophila melanogaster females.

Diet plays a significant role in maintaining lifelong health. In particular, lowering the dietary protein: carbohydrate ratio can improve lifespan. This has been interpreted as a direct effect of these macronutrients on physiology. Using Drosophila melanogaster, we show that the role of protein and carbohydrate on lifespan is indirect, acting by altering the partitioning of limiting amounts of dietary sterols between reproduction and lifespan. Shorter lifespans in flies fed on high protein: carbohydrate diets can be rescued by supplementing their food with cholesterol. Not only does this fundamentally alter the way we interpret the mechanisms of lifespan extension by dietary restriction, these data highlight the important principle that life histories can be affected by nutrient-dependent trade-offs that are indirect and independent of the nutrients (often macronutrients) that are the focus of study. This brings us closer to understanding the mechanistic basis of dietary restriction.

For the past fifteen years, animal studies have consistently shown that a low-protein, high-carbohydrate ('carbs') diet can extend the lifespan of many organisms, but at the cost of the number of offspring an individual can produce. Yet, it is still unclear what the best dietary balance is, and how these effects arise. One potential explanation could be that reproduction damages the body: low levels of proteins would therefore prolong life by lowering the reproductive output. Here, Zanco et al. examined the possibility that protein intake in fruit flies could instead be acting indirectly by changing the levels of a fat-like molecule called cholesterol, which is used to maintain the body and to support reproduction. To test this idea, groups of fruit flies were fed high levels of proteins. This led to increased reproduction rates, in turn depleting the mothers' reserves of cholesterol. Without enough of the molecule in their diet, the insects were less able to maintain their bodies, which reduced their lifespan. When Zanco et al. added cholesterol to a high-protein diet, the flies lived for the normal length of time. Longer lifespan therefore did not require restriction of the diet or any of its components. In fact, the flies that lived the longest ate protein rich diets, and reproduced the most. This study helps to better understand why changes in diet can influence how long an organism lives for, highlighting that the abundance of certain key molecules may be more important than restricting the levels of proteins, carbs or calories actually consumed.