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.

Sex-specific transgenerational effects of diet on offspring life history and physiology.

Dietary variation in males and females can shape the expression of offspring life histories and physiology. However, the relative contributions of maternal and paternal dietary variation to phenotypic expression of latter generations is currently unknown. We provided male and female Drosophila melanogaster grandparents with diets differing in sucrose concentration prior to reproduction, and similarly subjected their grandoffspring to the same treatments. We then investigated the phenotypic consequences of this dietary variation among the grandsons and granddaughters. We observed transgenerational effects of dietary sucrose, mediated through the grandmaternal lineage, which mimic the direct effects of sucrose on lifespan, with opposing patterns across sexes; low sucrose increased female, but decreased male, lifespan. Dietary mismatching of grandoffspring-grandparent diets increased lifespan and reproductive success, and moderated triglyceride levels of grandoffspring, providing insights into the physiological underpinnings of the complex transgenerational effects on life histories.

Wireless, intraoral hybrid electronics for real-time quantification of sodium intake toward hypertension management.

Recent wearable devices offer portable monitoring of biopotentials, heart rate, or physical activity, allowing for active management of human health and wellness. Such systems can be inserted in the oral cavity for measuring food intake in regard to controlling eating behavior, directly related to diseases such as hypertension, diabetes, and obesity. However, existing devices using plastic circuit boards and rigid sensors are not ideal for oral insertion. A user-comfortable system for the oral cavity requires an ultrathin, low-profile, and soft electronic platform along with miniaturized sensors. Here, we introduce a stretchable hybrid electronic system that has an exceptionally small form factor, enabling a long-range wireless monitoring of sodium intake. Computational study of flexible mechanics and soft materials provides fundamental aspects of key design factors for a tissue-friendly configuration, incorporating a stretchable circuit and sensor. Analytical calculation and experimental study enables reliable wireless circuitry that accommodates dynamic mechanical stress. Systematic in vitro modeling characterizes the functionality of a sodium sensor in the electronics. In vivo demonstration with human subjects captures the device feasibility for real-time quantification of sodium intake, which can be used to manage hypertension.

Commensal bacteria and essential amino acids control food choice behavior and reproduction.

Choosing the right nutrients to consume is essential to health and wellbeing across species. However, the factors that influence these decisions are poorly understood. This is particularly true for dietary proteins, which are important determinants of lifespan and reproduction. We show that in Drosophila melanogaster, essential amino acids (eAAs) and the concerted action of the commensal bacteria Acetobacter pomorum and Lactobacilli are critical modulators of food choice. Using a chemically defined diet, we show that the absence of any single eAA from the diet is sufficient to elicit specific appetites for amino acid (AA)-rich food. Furthermore, commensal bacteria buffer the animal from the lack of dietary eAAs: both increased yeast appetite and decreased reproduction induced by eAA deprivation are rescued by the presence of commensals. Surprisingly, these effects do not seem to be due to changes in AA titers, suggesting that gut bacteria act through a different mechanism to change behavior and reproduction. Thus, eAAs and commensal bacteria are potent modulators of feeding decisions and reproductive output. This demonstrates how the interaction of specific nutrients with the microbiome can shape behavioral decisions and life history traits.

Neurodynamics of time consciousness: An extensionalist explanation of apparent motion and the specious present via reentrant oscillatory multiplexing.

This paper situates an original model of reentrant oscillatory multiplexing within the philosophy of time consciousness to argue for an extensionalist theory of the specious present. I develop a detailed differential latency model of apparent motion to show how the ordinality of experiential content is isomorphic to the ordinality of relevant brain processes. I argue that the theory presented has resources to account for other key features of the specious present, including the representational discreteness between successive conscious moments as well as the phenomenological continuity between them. This work not only shows the plausibility of an extensionalist philosophical theory, it also illustrates the utility of differential latency views in squaring temporal illusions with empirically supported neurodynamics.

Nuclear hormone receptor DHR96 mediates the resistance to xenobiotics but not the increased lifespan of insulin-mutant Drosophila.

Lifespan of laboratory animals can be increased by genetic, pharmacological, and dietary interventions. Increased expression of genes involved in xenobiotic metabolism, together with resistance to xenobiotics, are frequent correlates of lifespan extension in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila, and mice. The Green Theory of Aging suggests that this association is causal, with the ability of cells to rid themselves of lipophilic toxins limiting normal lifespan. To test this idea, we experimentally increased resistance of Drosophila to the xenobiotic dichlordiphenyltrichlorethan (DDT), by artificial selection or by transgenic expression of a gene encoding a cytochrome P450. Although both interventions increased DDT resistance, neither increased lifespan. Furthermore, dietary restriction increased lifespan without increasing xenobiotic resistance, confirming that the two traits can be uncoupled. Reduced activity of the insulin/Igf signaling (IIS) pathway increases resistance to xenobiotics and extends lifespan in Drosophila, and can also increase longevity in C. elegans, mice, and possibly humans. We identified a nuclear hormone receptor, DHR96, as an essential mediator of the increased xenobiotic resistance of IIS mutant flies. However, the IIS mutants remained long-lived in the absence of DHR96 and the xenobiotic resistance that it conferred. Thus, in Drosophila IIS mutants, increased xenobiotic resistance and enhanced longevity are not causally connected. The frequent co-occurrence of the two traits may instead have evolved because, in nature, lowered IIS can signal the presence of pathogens. It will be important to determine whether enhanced xenobiotic metabolism is also a correlated, rather than a causal, trait in long-lived mice.

Drosophila as a model for ageing.

Drosophila melanogaster has been a key model in developing our current understanding of the molecular mechanisms of ageing. Of particular note is its role in establishing the evolutionary conservation of reduced insulin and IGF-1-like signaling in promoting healthy ageing. Capitalizing on its many advantages for experimentation, more recent work has revealed how precise nutritional and genetic interventions can improve fly lifespan without obvious detrimental side effects. We give a brief summary of these recent findings as well as examples of how they may modify ageing via actions in the gut and muscle. These discoveries highlight how expanding our understanding of metabolic and signaling interconnections will provide even greater insight into how these benefits may be harnessed for anti-ageing interventions.

Using Mouse and Drosophila Models to Investigate the Mechanistic Links between Diet, Obesity, Type II Diabetes, and Cancer.

Many of the links between diet and cancer are controversial and over simplified. To date, human epidemiological studies consistently reveal that patients who suffer diet-related obesity and/or type II diabetes have an increased risk of cancer, suffer more aggressive cancers, and respond poorly to current therapies. However, the underlying molecular mechanisms that increase cancer risk and decrease the response to cancer therapies in these patients remain largely unknown. Here, we review studies in mouse cancer models in which either dietary or genetic manipulation has been used to model obesity and/or type II diabetes. These studies demonstrate an emerging role for the conserved insulin and insulin-like growth factor signaling pathways as links between diet and cancer progression. However, these models are time consuming to develop and expensive to maintain. As the world faces an epidemic of obesity and type II diabetes we argue that the development of novel animal models is urgently required. We make the case for Drosophila as providing an unparalleled opportunity to combine dietary manipulation with models of human metabolic disease and cancer. Thus, combining diet and cancer models in Drosophila can rapidly and significantly advance our understanding of the conserved molecular mechanisms that link diet and diet-related metabolic disorders to poor cancer patient prognosis.

Sex and genotype effects on nutrient-dependent fitness landscapes in Drosophila melanogaster.

The sexes perform different reproductive roles and have evolved sometimes strikingly different phenotypes. One focal point of adaptive divergence occurs in the context of diet and metabolism, and males and females of a range of species have been shown to require different nutrients to maximize their fitness. Biochemical analyses in Drosophila melanogaster have confirmed that dimorphism in dietary requirements is associated with molecular sex differences in metabolite titres. In addition, they also showed significant within-sex genetic variation in the metabolome. To date however, it is unknown whether this metabolic variation translates into differences in reproductive fitness. The answer to this question is crucial to establish whether genetic variation is selectively neutral or indicative of constraints on sex-specific physiological adaptation and optimization. Here we assay genetic variation in consumption and metabolic fitness effects by screening male and female fitness of thirty D. melanogaster genotypes across four protein-to-carbohydrate ratios. In addition to confirming sexual dimorphism in consumption and fitness, we find significant genetic variation in male and female dietary requirements. Importantly, these differences are not explained by feeding responses and probably reflect metabolic variation that, in turn, suggests the presence of genetic constraints on metabolic dimorphism.

A holidic medium for Drosophila melanogaster.

A critical requirement for research using model organisms is a well-defined and consistent diet. There is currently no complete chemically defined (holidic) diet available for Drosophila melanogaster. We describe a holidic medium that is equal in performance to an oligidic diet optimized for adult fecundity and lifespan. This holidic diet supports development over multiple generations but at a reduced rate. Over 7 years of experiments, the holidic diet yielded more consistent experimental outcomes than did oligidic food for egg laying by females. Nutrients and drugs were more available to flies in holidic medium and, similar to dietary restriction on oligidic food, amino acid dilution increased fly lifespan. We used this holidic medium to investigate amino acid-specific effects on food-choice behavior and report that folic acid from the microbiota is sufficient for Drosophila development.

Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila.

Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.

Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila.

Dietary restriction extends healthy lifespan in diverse organisms and reduces fecundity. It is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature. If this were the case, long life under dietary restriction and high fecundity under full feeding would be mutually exclusive, through competition for the same limiting nutrients. Here we report a test of this idea in which we identified the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan, similar to the effects of full feeding, with other nutrients having little or no effect. However, methionine alone was necessary and sufficient to increase fecundity as much as did full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the responses to dietary restriction. Lifespan was decreased by the addition of amino acids, with an interaction between methionine and other essential amino acids having a key role. Hence, an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during dietary restriction. Reduced activity of the insulin/insulin-like growth factor signalling pathway extends lifespan in diverse organisms, and we find that it also protects against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits to lifespan of dietary restriction without incurring a reduction in fecundity, through a suitable balance of nutrients in the diet.

A Simple Method for Quantifying Larval Locomotion in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is a powerful genetic model that has been used for many decades to study nervous system function, development, and behavior. There are a large number of developmental and behavioral traits that can be measured to provide a broad readout of neurological function. These include patterned motor behaviors, such as larval locomotion, which can be used to assess whether genetic or environmental factors affect nervous system function to provide an entry point for deeper mechanistic studies. Here, we describe a protocol for quantifying larval locomotion using a simple camera setup and a freely available image analysis software. This protocol can be readily applied to human disease models or in toxicology studies, for example, to broadly assess the impact of treatments on neurological function.

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.

Identifying potential dietary treatments for inherited metabolic disorders using Drosophila nutrigenomics.

Inherited metabolic disorders are a group of genetic conditions that can cause severe neurological impairment and child mortality. Uniquely, these disorders respond to dietary treatment; however, this option remains largely unexplored because of low disorder prevalence and the lack of a suitable paradigm for testing diets. Here, we screened 35 Drosophila amino acid disorder models for disease-diet interactions and found 26 with diet-altered development and/or survival. Using a targeted multi-nutrient array, we examine the interaction in a model of isolated sulfite oxidase deficiency, an infant-lethal disorder. We show that dietary cysteine depletion normalizes their metabolic profile and rescues development, neurophysiology, behavior, and lifelong fly survival, thus providing a basis for further study into the pathogenic mechanisms involved in this disorder. Our work highlights the diet-sensitive nature of metabolic disorders and establishes Drosophila as a valuable tool for nutrigenomic studies for informing potential dietary therapies.

A defined diet for pre-adult Drosophila melanogaster.

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that reduces the developmental period, restores fat levels, enhances body mass, and fully rescues survivorship without compromise to adult lifespan. To demonstrate an application of this formula, we explored pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder affecting the metabolism of branched-chain amino acids. We reveal rapid, specific, and predictable nutrient effects on the disease state consistent with observations from mouse and patient studies. Together, our diet provides a powerful means with which to examine the interplay between diet and metabolism across all life stages in an animal model.

Exome-informed formulations of food proteins enhance body growth and feed conversion efficiency in ad libitum-fed mice.

Meeting requirements for dietary proteins, especially of essential amino acids (EAAs), is critical for the life-long health of living organisms. However, defining EAA targets for preparing biologically-matched nutrition that satisfies metabolic requirements for protein remains challenging. Previous research has shown the advantages of 'exome matching' in representing the specific requirement of dietary AAs, where the target dietary AA profile was derived from in silico translation of the genome of an organism, specifically responsible for protein expression (the 'exome'). However, past studies have assessed these effects in only one sex, for few parameters (body mass and composition), and have used purified diets in which protein is supplied as a mixture of individual AAs. Here, for the first time, we utilise a computational method to guide the formulation of custom protein blends and test if exome matching can be achieved at the intact protein level, through blending standard protein ingredients, ultimately leading to optimal growth, longevity and reproductive function. Mice were provided ad libitum (ad lib) access to one of the four iso-energetic protein-limited diets, two matched and two mis-matched to the mouse exome target, and fed at a fixed protein energy level of 6.2%. During or following 13-weeks of feeding, the food intake, body growth, composition and reproductive functions were measured. Compared to the two mis-matched diets, male and female animals on the exome-matched diet with protein digestibility correction applied, exhibited significantly improved growth rates and final body mass. The feed conversion efficiency in the same diet was also increased by 62% and 40% over the worst diets for males and females, respectively. Male, not female, exhibited higher accretion of lean body mass with the matched, digestibility-corrected diet. All reproductive function measures in both sexes were comparable among diets, with the exception of testicular daily sperm production in males, which was higher in the two matched diets versus the mis-matched diets. The results collectively demonstrate the pronounced advantages of exome-matching in supporting body growth and improving feed conversion efficiency in both sexes. However, the potential impact of this approach in enhancing fertility needs further investigation.

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.

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.

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.