Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities.

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

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.

Developmental neuroscience: Building sex-specific adult circuitry from common larval origins.

The development of sex-specific neural circuitry is critical for reproductive behaviors. A new study traces the developmental origin of female-specific neurons that underlie an adult mating behavior to larval neurons common to both sexes in Drosophila.

Machine learning reveals the control mechanics of an insect wing hinge.

Insects constitute the most species-rich radiation of metazoa, a success that is due to the evolution of active flight. Unlike pterosaurs, birds and bats, the wings of insects did not evolve from legs1, but are novel structures that are attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings2. The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the three-dimensional motion of the wings with high-speed cameras. Using machine learning, we created a convolutional neural network3 that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder4 that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation incorporating our hinge model generates flight manoeuvres that are remarkably similar to those of free-flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila.

Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. We previously discovered that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust (Hampel et al., 2017; Seeds et al., 2014). Here, we identify nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

An open platform for visual stimulation of insects.

To study how the nervous system processes visual information, experimenters must record neural activity while delivering visual stimuli in a controlled fashion. In animals with a nearly panoramic field of view, such as flies, precise stimulation of the entire visual field is challenging. We describe a projector-based device for stimulation of the insect visual system under a microscope. The device is based on a bowl-shaped screen that provides a wide and nearly distortion-free field of view. It is compact, cheap, easy to assemble, and easy to operate using the included open-source software for stimulus generation. We validate the virtual reality system technically and demonstrate its capabilities in a series of experiments at two levels: the cellular, by measuring the membrane potential responses of visual interneurons; and the organismal, by recording optomotor and fixation behavior of Drosophila melanogaster in tethered flight. Our experiments reveal the importance of stimulating the visual system of an insect with a wide field of view, and we provide a simple solution to do so.

Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

Octopamine integrates the status of internal energy supply into the formation of food-related memories.

The brain regulates food intake in response to internal energy demands and food availability. However, can internal energy storage influence the type of memory that is formed? We show that the duration of starvation determines whether Drosophila melanogaster forms appetitive short-term or longer-lasting intermediate memories. The internal glycogen storage in the muscles and adipose tissue influences how intensely sucrose-associated information is stored. Insulin-like signaling in octopaminergic reward neurons integrates internal energy storage into memory formation. Octopamine, in turn, suppresses the formation of long-term memory. Octopamine is not required for short-term memory because octopamine-deficient mutants can form appetitive short-term memory for sucrose and to other nutrients depending on the internal energy status. The reduced positive reinforcing effect of sucrose at high internal glycogen levels, combined with the increased stability of food-related memories due to prolonged periods of starvation, could lead to increased food intake.

Deciding what and how much to eat is a complex biological process which involves balancing many types of information such as the levels of internal energy storage, the amount of food previously available in the environment, the perceived value of certain food items, and how these are remembered. At the molecular level, food contains carbohydrates that are broken down to produce glucose, which is then delivered to cells under the control of a hormone called insulin. There, glucose molecules are either immediately used or stored as glycogen until needed. Insulin signalling is also known to interact with the brain's decision-making systems that control eating behaviors; however, how our brains balance food intake with energy storage is poorly understood. Berger et al. set out to investigate this question using fruit flies as an experimental model. These insects also produce insulin-like molecules which help to relay information about glycogen levels to the brain's decision-making system. In particular, these signals reach a population of neurons that produce a messenger known as octopamine similar to the human noradrenaline, which helps regulate how much the flies find consuming certain types of foods rewarding. Berger et al. were able to investigate the role of octopamine in helping to integrate information about internal and external resource levels, memory formation and the evaluation of different food types. When the insects were fed normally, increased glycogen levels led to foods rich in carbohydrates being rated as less rewarding by the decision-making cells, and therefore being consumed less. Memories related to food intake were also short-lived ­ in other words, long-term 'food memory' was suppressed, re-setting the whole system after every meal. In contrast, long periods of starvation in insects with high carbohydrates resources produced a stable, long-term memory of food and hunger which persisted even after the flies had fed again. This experience also changed their food rating system, with highly nutritious foods no longer being perceived as sufficiently rewarding. As a result, the flies overate. This study sheds new light on the mechanisms our bodies may use to maintain energy reserves when food is limited. The persistence of 'food memory' after long periods of starvation may also explain why losing weight is difficult, especially during restrictive diets. In the future, Berger et al. hope that this knowledge will contribute to better strategies for weight management.

Mitochondrial background can explain variable costs of immune deployment.

Organismal health and survival depend on the ability to mount an effective immune response against infection. Yet immune defence may be energy-demanding, resulting in fitness costs if investment in immune function deprives other physiological processes of resources. While evidence of costly immunity resulting in reduced longevity and reproduction is common, the role of energy-producing mitochondria on the magnitude of these costs is unknown. Here we employed Drosophila melanogaster cybrid lines, where several mitochondrial genotypes (mitotypes) were introgressed onto a single nuclear genetic background, to explicitly test the role of mitochondrial variation on the costs of immune stimulation. We exposed female flies carrying one of nine distinct mitotypes to either a benign, heat-killed bacterial pathogen (stimulating immune deployment while avoiding pathology) or a sterile control and measured lifespan, fecundity, and locomotor activity. We observed mitotype-specific costs of immune stimulation and identified a positive genetic correlation between life span and the proportion of time cybrids spent moving while alive. Our results suggest that costs of immunity are highly variable depending on the mitochondrial genome, adding to a growing body of work highlighting the important role of mitochondrial variation in host-pathogen interactions.

Integrated stress response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the integrated stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor that instructs oogenesis. We demonstrate that Atf4 regulates lipase activity to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient-sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat-body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction-directly by impacting yolk lipoprotein production and follicle maturation and systemically by regulating ovulation.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

Motor neurons generate pose-targeted movements via proprioceptive sculpting.

Motor neurons are the final common pathway1 through which the brain controls movement of the body, forming the basic elements from which all movement is composed. Yet how a single motor neuron contributes to control during natural movement remains unclear. Here we anatomically and functionally characterize the individual roles of the motor neurons that control head movement in the fly, Drosophila melanogaster. Counterintuitively, we find that activity in a single motor neuron rotates the head in different directions, depending on the starting posture of the head, such that the head converges towards a pose determined by the identity of the stimulated motor neuron. A feedback model predicts that this convergent behaviour results from motor neuron drive interacting with proprioceptive feedback. We identify and genetically2 suppress a single class of proprioceptive neuron3 that changes the motor neuron-induced convergence as predicted by the feedback model. These data suggest a framework for how the brain controls movements: instead of directly generating movement in a given direction by activating a fixed set of motor neurons, the brain controls movements by adding bias to a continuing proprioceptive-motor loop.

To be or not to be: orb, the fusome and oocyte specification in Drosophila.

In the fruit fly Drosophila melanogaster, two cells in a cyst of 16 interconnected cells have the potential to become the oocyte, but only one of these will assume an oocyte fate as the cysts transition through regions 2a and 2b of the germarium. The mechanism of specification depends on a polarized microtubule network, a dynein dependent Egl:BicD mRNA cargo complex, a special membranous structure called the fusome and its associated proteins, and the translational regulator orb. In this work, we have investigated the role of orb and the fusome in oocyte specification. We show here that specification is a stepwise process. Initially, orb mRNAs accumulate in the two pro-oocytes in close association with the fusome. This association is accompanied by the activation of the orb autoregulatory loop, generating high levels of Orb. Subsequently, orb mRNAs become enriched in only one of the pro-oocytes, the presumptive oocyte, and this is followed, with a delay, by Orb localization to the oocyte. We find that fusome association of orb mRNAs is essential for oocyte specification in the germarium, is mediated by the orb 3' UTR, and requires Orb protein. We also show that the microtubule minus end binding protein Patronin functions downstream of orb in oocyte specification. Finally, in contrast to a previously proposed model for oocyte selection, we find that the choice of which pro-oocyte becomes the oocyte does not seem to be predetermined by the amount of fusome material in these two cells, but instead depends upon a competition for orb gene products.

Transforming a head direction signal into a goal-oriented steering command.

To navigate, we must continuously estimate the direction we are headed in, and we must correct deviations from our goal1. Direction estimation is accomplished by ring attractor networks in the head direction system2,3. However, we do not fully understand how the sense of direction is used to guide action. Drosophila connectome analyses4,5 reveal three cell populations (PFL3R, PFL3L and PFL2) that connect the head direction system to the locomotor system. Here we use imaging, electrophysiology and chemogenetic stimulation during navigation to show how these populations function. Each population receives a shifted copy of the head direction vector, such that their three reference frames are shifted approximately 120° relative to each other. Each cell type then compares its own head direction vector with a common goal vector; specifically, it evaluates the congruence of these vectors via a nonlinear transformation. The output of all three cell populations is then combined to generate locomotor commands. PFL3R cells are recruited when the fly is oriented to the left of its goal, and their activity drives rightward turning; the reverse is true for PFL3L. Meanwhile, PFL2 cells increase steering speed, and are recruited when the fly is oriented far from its goal. PFL2 cells adaptively increase the strength of steering as directional error increases, effectively managing the tradeoff between speed and accuracy. Together, our results show how a map of space in the brain can be combined with an internal goal to generate action commands, via a transformation from world-centric coordinates to body-centric coordinates.

Structural basis for sugar perception by Drosophila gustatory receptors.

Insects rely on a family of seven transmembrane proteins called gustatory receptors (GRs) to encode different taste modalities, such as sweet and bitter. We report structures of Drosophila sweet taste receptors GR43a and GR64a in the apo and sugar-bound states. Both GRs form tetrameric sugar-gated cation channels composed of one central pore domain (PD) and four peripheral ligand-binding domains (LBDs). Whereas GR43a is specifically activated by the monosaccharide fructose that binds to a narrow pocket in LBDs, disaccharides sucrose and maltose selectively activate GR64a by binding to a larger and flatter pocket in LBDs. Sugar binding to LBDs induces local conformational changes, which are subsequently transferred to the PD to cause channel opening. Our studies reveal a structural basis for sugar recognition and activation of GRs.

Endurance exercise upregulates mtp expression in aged Drosophila to ameliorate age-related diastolic dysfunction and extend lifespan.

Diastolic dysfunction is a major cardiac dysfunction, and an important predisposing factor is age. Although exercise training is often used for the prevention and treatment of cardiovascular disease nowadays, little is currently known about whether exercise interventions associated with the slowing of cardiac aging are related to mtp-related pathways. In the present study, the UAS/Tub-Gal4 system was used to knockdown whole-body mtp expression levels in Drosophila, which underwent 2 weeks of endurance training. By conducting different assays and quantifying different indicators, we sought to investigate the relationship between mtp, exercise, and age-related diastolic dysfunction. We found that (1) Drosophila in the mtpRNAi youth group exhibited age-related diastolic dysfunction and had a significantly shorter mean lifespan. (2) Endurance exercise could improve diastolic dysfunction and prolong lifespan in aged Drosophila. (3) Endurance exercise could increase the expression levels of apolpp and Acox3, and decrease the levels of TC, LDL-C, and TG in the aged group. In summary, aging causes age-associated diastolic dysfunction in Drosophila, and systemic knockdown of mtp causes premature age-associated diastolic dysfunction in young Drosophila. Besides, endurance exercise improves age-related diastolic dysfunction and prolongs lifespan.

Betulinic Acid Increases the Lifespan of Drosophila melanogaster via Sir2 and FoxO Activation.

Betulinic acid (BetA), a triterpenoid derivative found abundantly in the plant kingdom, has emerged as a promising candidate for promoting longevity. Many research studies have shown its antioxidant, anti-inflammatory, antiviral, and anticancer activities, making it an interesting subject for investigating its potential influence on lifespan. This study aimed to investigate the effects of BetA on longevity and the mechanisms associated with it using the fruit fly Drosophila melanogaster as the organism model. The results showed that 50 µM BetA supplementation extended the mean lifespan of fruit flies by 13% in males and 6% in females without any adverse effects on their physiology, such as fecundity, feeding rate, or locomotion ability reduction. However, 50 µM BetA supplementation failed to increase the lifespan in mutants lacking functional silent information regulator 2 (Sir2) and Forkhead box O (FoxO)-null, implying that the longevity effect of BetA is related to Sir2 and FoxO activation. Our study contributes to the knowledge in the field of anti-aging research and inspires further investigations into natural compounds such as BetA to enhance organismal healthspan.

Converting an allocentric goal into an egocentric steering signal.

Neuronal signals that are relevant for spatial navigation have been described in many species1-10. However, a circuit-level understanding of how such signals interact to guide navigational behaviour is lacking. Here we characterize a neuronal circuit in the Drosophila central complex that compares internally generated estimates of the heading and goal angles of the fly-both of which are encoded in world-centred (allocentric) coordinates-to generate a body-centred (egocentric) steering signal. Past work has suggested that the activity of EPG neurons represents the fly's moment-to-moment angular orientation, or heading angle, during navigation2,11. An animal's moment-to-moment heading angle, however, is not always aligned with its goal angle-that is, the allocentric direction in which it wishes to progress forward. We describe FC2 cells12, a second set of neurons in the Drosophila brain with activity that correlates with the fly's goal angle. Focal optogenetic activation of FC2 neurons induces flies to orient along experimenter-defined directions as they walk forward. EPG and FC2 neurons connect monosynaptically to a third neuronal class, PFL3 cells12,13. We found that individual PFL3 cells show conjunctive, spike-rate tuning to both the heading angle and the goal angle during goal-directed navigation. Informed by the anatomy and physiology of these three cell classes, we develop a model that explains how this circuit compares allocentric heading and goal angles to build an egocentric steering signal in the PFL3 output terminals. Quantitative analyses and optogenetic manipulations of PFL3 activity support the model. Finally, using a new navigational memory task, we show that flies expressing disruptors of synaptic transmission in subsets of PFL3 cells have a reduced ability to orient along arbitrary goal directions, with an effect size in quantitative accordance with the prediction of our model. The biological circuit described here reveals how two population-level allocentric signals are compared in the brain to produce an egocentric output signal that is appropriate for motor control.

Developmental remodeling repurposes larval neurons for sexual behaviors in adult Drosophila.

Most larval neurons in Drosophila are repurposed during metamorphosis for functions in adult life, but their contribution to the neural circuits for sexually dimorphic behaviors is unknown. Here, we identify two interneurons in the nerve cord of adult Drosophila females that control ovipositor extrusion, a courtship rejection behavior performed by mated females. We show that these two neurons are present in the nerve cord of larvae as mature, sexually monomorphic interneurons. During pupal development, they acquire the expression of the sexual differentiation gene, doublesex; undergo doublesex-dependent programmed cell death in males; and are remodeled in females for functions in female mating behavior. Our results demonstrate that the neural circuits for courtship in Drosophila are built in part using neurons that are sexually reprogrammed from former sex-shared activities in larval life.