Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila.

Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.

Large-language models facilitate discovery of the molecular signatures regulating sleep and activity.

Sleep, locomotor and social activities are essential animal behaviors, but their reciprocal relationships and underlying mechanisms remain poorly understood. Here, we elicit information from a cutting-edge large-language model (LLM), generative pre-trained transformer (GPT) 3.5, which interprets 10.2-13.8% of Drosophila genes known to regulate the 3 behaviors. We develop an instrument for simultaneous video tracking of multiple moving objects, and conduct a genome-wide screen. We have identified 758 fly genes that regulate sleep and activities, including mre11 which regulates sleep only in the presence of conspecifics, and NELF-B which regulates sleep regardless of whether conspecifics are present. Based on LLM-reasoning, an educated signal web is modeled for understanding of potential relationships between its components, presenting comprehensive molecular signatures that control sleep, locomotor and social activities. This LLM-aided strategy may also be helpful for addressing other complex scientific questions.

Naturally occurring viruses of Drosophila reduce offspring number and lifespan.

Drosophila remains a pre-eminent insect model system for host-virus interaction, but the host range and fitness consequences of the drosophilid virome are poorly understood. Metagenomic studies have reported approximately 200 viruses associated with Drosophilidae, but few isolates are available to characterize the Drosophila immune response, and most characterization has relied on injection and systemic infection. Here, we use a more natural infection route to characterize the fitness effects of infection and to study a wider range of viruses. We exposed laboratory Drosophila melanogaster to 23 naturally occurring viruses from wild-collected drosophilids. We recorded transmission rates along with two components of female fitness: survival and the lifetime number of adult offspring produced. Nine different viruses transmitted during contact with laboratory D. melanogaster, although for the majority, rates of transmission were less than 20%. Five virus infections led to a significant decrease in lifespan (D. melanogaster Nora virus, D. immigrans Nora virus, Muthill virus, galbut virus and Prestney Burn virus), and three led to a reduction in the total number of offspring. Our findings demonstrate the utility of the Drosophila model for community-level studies of host-virus interactions, and suggest that viral infection could be a substantial fitness burden on wild flies.

Alpha-ketoglutarate supplementation in long-lived Drosophila melanogaster: Impact on lifespan and metabolic responses.

Studies on antiaging remedies in insect models sometimes show discrepancies in results. These discrepancies could be explained by different responses of short- and long-lived strains on the antiaging remedies. The purpose of the study was to test whether life-prolonging effects of alpha-ketoglutarate (AKG), observed in nematodes and fruit flies, would be reproduced in long-lived Drosophila melanogaster flies. Lifespan was assayed in flies kept in demographic cages. Fecundity, proportion of flies capable of negative geotaxis, starvation resistance, time of heat coma onset, levels of triacyglycerols, body glucose, glycogen, activities of glutamate dehydrogenase, catalase, glutathione-S-transferase, hexokinase, phosphofructokinase, pyruvate kinase, lactate, and glutamate dehydrogenases were assessed. Dietary AKG did not affect fly lifespan on the diet with 5% yeast and 5% sucrose (5Y:5S) and on the diet with 9% yeast and 1% sucrose (9Y:1S), but increased lifespan on the low-protein diet (1Y:9S). Twenty-five-day-old female flies fed a 5Y:5S diet with 10 mM AKG for 3 weeks, did not differ from the control group (without AKG) in climbing activity, resistance to heat stress, and starvation. The levels of glucose and glycogen were unaffected but the levels of triacylglycerols were lower in AKG-fed female flies. No differences in activities of glycolytic enzymes, NADPH-producing enzymes, glutamate dehydrogenase, oxygen consumption, and levels of oxidative stress markers were observed between the control and AKG-fed flies. However, AKG-fed flies had lower activities of catalase and glutathione-S-transferase. These results suggest that potential antiaging remedies, such as AKG, may not extend lifespan in long-living organisms despite influencing several metabolic parameters.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods: We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results: We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions: Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Sexual Selection Increases Male Behavioral Consistency in Drosophila melanogaster.

AbstractSexual selection has been suggested to influence the expression of male behavioral consistency. However, despite predictions, direct experimental support for this hypothesis has been lacking. Here, we investigated whether sexual selection altered male behavioral consistency in Drosophila melanogaster-a species with both pre- and postcopulatory sexual selection. We took 1,144 measures of locomotor activity (a fitness-related trait in D. melanogaster) from 286 flies derived from replicated populations that have experimentally evolved under either high or low levels of sexual selection for >320 generations. We found that high sexual selection males were more consistent (decreased within-individual variance) in their locomotor activity than male conspecifics from low sexual selection populations. There were no differences in behavioral consistency between females from the high and low sexual selection populations. Furthermore, while females were more behaviorally consistent than males in the low sexual selection populations, there were no sex differences in behavioral consistency in high sexual selection populations. Our results demonstrate that behavioral plasticity is reduced in males from populations exposed to high levels of sexual selection. Disentangling whether these effects represent an evolved response to changes in the intensity of selection or are manifested through nongenetic parental effects represents a challenge for future research.

Theoretical principles explain the structure of the insect head direction circuit.

To navigate their environment, insects need to keep track of their orientation. Previous work has shown that insects encode their head direction as a sinusoidal activity pattern around a ring of neurons arranged in an eight-column structure. However, it is unclear whether this sinusoidal encoding of head direction is just an evolutionary coincidence or if it offers a particular functional advantage. To address this question, we establish the basic mathematical requirements for direction encoding and show that it can be performed by many circuits, all with different activity patterns. Among these activity patterns, we prove that the sinusoidal one is the most noise-resilient, but only when coupled with a sinusoidal connectivity pattern between the encoding neurons. We compare this predicted optimal connectivity pattern with anatomical data from the head direction circuits of the locust and the fruit fly, finding that our theory agrees with experimental evidence. Furthermore, we demonstrate that our predicted circuit can emerge using Hebbian plasticity, implying that the neural connectivity does not need to be explicitly encoded in the genetic program of the insect but rather can emerge during development. Finally, we illustrate that in our theory, the consistent presence of the eight-column organisation of head direction circuits across multiple insect species is not a chance artefact but instead can be explained by basic evolutionary principles.

Insects, including fruit flies and locusts, move throughout their environment to find food, interact with each other or escape danger. To navigate their surroundings, insects need to be able to keep track of their orientation. This tracking is achieved through visual cues and integrating information about their movements whilst flying so they know which direction their head is facing. The set of neurons responsible for relaying information about the direction of the head (also known as heading) are connected together in a ring made up of eight columns of cells. Previous studies showed that the level of activity across this ring of neurons resembles a sinusoid shape: a smooth curve with one peak which encodes the animal's heading. Neurons downstream from this eight-column ring, which relay velocity information, also display this sinusoidal pattern of activation. Aceituno, Dall'Osto and Pisokas wanted to understand whether this sinusoidal pattern was an evolutionary coincidence, or whether it offers a particular advantage to insects. To answer this question, they established the mathematical criteria required for neurons in the eight-column ring to encode information about the heading of the animal. This revealed that these conditions can be satisfied by many different patterns of activation, not just the sinusoidal shape. However, Aceituno, Dall'Osto and Pisokas show that the sinusoidal shape is the most resilient to variations in neuronal activity which may impact the encoded information. Further experiments revealed that this resilience only occurred if neurons in the circuit were connected together in a certain pattern. Aceituno, Dall'Osto and Pisokas then compared this circuit with experimental data from locusts and fruit flies and found that both insects exhibit the predicted connection pattern. They also discovered that animals do not have to be born with this neuronal connection pattern, but can develop it during their lifetime. These findings provide fresh insights into how insects relay information about the direction of their head as they fly. They suggest that the structure of the neuronal circuit responsible for encoding head direction was not formed by chance but instead arose due to the evolutionary benefits it provided.

Peripheral preprocessing in Drosophila facilitates odor classification.

The mammalian brain implements sophisticated sensory processing algorithms along multilayered ("deep") neural networks. Strategies that insects use to meet similar computational demands, while relying on smaller nervous systems with shallow architectures, remain elusive. Using Drosophila as a model, we uncover the algorithmic role of odor preprocessing by a shallow network of compartmentalized olfactory receptor neurons. Each compartment operates as a ratiometric unit for specific odor-mixtures. This computation arises from a simple mechanism: electrical coupling between two differently sized neurons. We demonstrate that downstream synaptic connectivity is shaped to optimally leverage amplification of a hedonic value signal in the periphery. Furthermore, peripheral preprocessing is shown to markedly improve novel odor classification in a higher brain center. Together, our work highlights a far-reaching functional role of the sensory periphery for downstream processing. By elucidating the implementation of powerful computations by a shallow network, we provide insights into general principles of efficient sensory processing algorithms.

Mapping model units to visual neurons reveals population code for social behaviour.

The rich variety of behaviours observed in animals arises through the interplay between sensory processing and motor control. To understand these sensorimotor transformations, it is useful to build models that predict not only neural responses to sensory input1-5 but also how each neuron causally contributes to behaviour6,7. Here we demonstrate a novel modelling approach to identify a one-to-one mapping between internal units in a deep neural network and real neurons by predicting the behavioural changes that arise from systematic perturbations of more than a dozen neuronal cell types. A key ingredient that we introduce is 'knockout training', which involves perturbing the network during training to match the perturbations of the real neurons during behavioural experiments. We apply this approach to model the sensorimotor transformations of Drosophila melanogaster males during a complex, visually guided social behaviour8-11. The visual projection neurons at the interface between the optic lobe and central brain form a set of discrete channels12, and prior work indicates that each channel encodes a specific visual feature to drive a particular behaviour13,14. Our model reaches a different conclusion: combinations of visual projection neurons, including those involved in non-social behaviours, drive male interactions with the female, forming a rich population code for behaviour. Overall, our framework consolidates behavioural effects elicited from various neural perturbations into a single, unified model, providing a map from stimulus to neuronal cell type to behaviour, and enabling future incorporation of wiring diagrams of the brain15 into the model.

The two autophagy-related proteins 8a and 8b play distinct physiological roles in Drosophila.

Atg8 family proteins play crucial roles in autophagy to maintain cellular homeostasis. However, the physiological roles of Atg8 family proteins have not been systematically determined. In this study, we generated Atg8a and Atg8b (homologs of Atg8 in Drosophila melanogaster) knockout flies. We found that the loss of Atg8a affected autophagy and resulted in partial lethality, abnormal wings, decreased lifespan, and decreased climbing ability in flies. Furthermore, the loss of Atg8a resulted in reduced muscle integrity and the progressive degeneration of the neuron system. We also found that the phosphorylation at Ser88 of Atg8a is important for autophagy and neuronal integrity. The loss of Atg8b did not affect autophagy but induced male sterility in flies. Here, we take full advantage of the fly system to elucidate the physiological function of Atg8a and Atg8b in Drosophila.

A subclass of evening cells promotes the switch from arousal to sleep at dusk.

Animals exhibit rhythmic patterns of behavior that are shaped by an internal circadian clock and the external environment. Although light intensity varies across the day, there are particularly robust differences at twilight (dawn/dusk). These periods are also associated with major changes in behavioral states, such as the transition from arousal to sleep. However, the neural mechanisms by which time and environmental conditions promote these behavioral transitions are poorly defined. Here, we show that the E1 subclass of Drosophila evening clock neurons promotes the transition from arousal to sleep at dusk. We first demonstrate that the cell-autonomous clocks of E2 neurons primarily drive and adjust the phase of evening anticipation, the canonical behavior associated with "evening" clock neurons. We next show that conditionally silencing E1 neurons causes a significant delay in sleep onset after dusk. However, rather than simply promoting sleep, activating E1 neurons produces time- and light-dependent effects on behavior. Activation of E1 neurons has no effect early in the day but then triggers arousal before dusk and induces sleep after dusk. Strikingly, these activation-induced phenotypes depend on the presence of light during the day. Despite their influence on behavior around dusk, in vivo voltage imaging of E1 neurons reveals that their spiking rate and pattern do not significantly change throughout the day. Moreover, E1-specific clock ablation has no effect on arousal or sleep. Thus, we suggest that, rather than specifying "evening" time, E1 neurons act, in concert with other rhythmic neurons, to promote behavioral transitions at dusk.

Preference of position in the proximity of various sugars revealed by location analysis of Drosophila melanogaster.

Feeding behaviors are determined by two main factors. One is the internal state, such as hunger or previous experiences; the other is external factors, such as sensory stimulation. During starvation, animals must balance food-seeking behavior with energy conservation. The fruit fly, Drosophila melanogaster, serves as a useful model for studying food selectivity and various behaviors related to food intake. However, few studies have directly connected food selectivity with other behaviors, such as locomotor activity and sleep. In this study, we report that flies exhibited a preference for specific positions and spent more time in the proximity of sweet sugars, such as sucrose and sucralose, but not non-sweet and nutritious sugars like xylitol and sorbitol. On the other hand, prolonged exposure to sorbitol increased the staying time of flies in the proximity of sorbitol. Additionally, after starvation, flies immediately exhibited a position preference in the proximity of sorbitol. These findings suggest that flies prefer the proximity of sweet food, and starvation alters their preference for nutritious food, which may be beneficial for their survival.

The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

Elevated sleep quota in a stress-resilient Drosophila species.

Sleep is broadly conserved across the animal kingdom but can vary widely between species. It is currently unclear which selective pressures and regulatory mechanisms influence differences in sleep between species. The fruit fly Drosophila melanogaster has become a successful model system for examining sleep regulation and function, but little is known about the sleep patterns in many related fly species. Here, we find that fly species with adaptations to extreme desert environments, including D. mojavensis, exhibit strong increases in baseline sleep compared with D. melanogaster. Long-sleeping D. mojavensis show intact homeostasis, indicating that desert flies carry an elevated drive for sleep. In addition, D. mojavensis exhibit altered abundance or distribution of several sleep/wake-related neuromodulators and neuropeptides that are consistent with their reduced locomotor activity and increased sleep. Finally, we find that in a nutrient-deprived environment, the sleep patterns of individual D. mojavensis are strongly correlated with their survival time and that disrupting sleep via constant light stimulation renders D. mojavensis more sensitive to starvation. Our results demonstrate that D. mojavensis is a novel model for studying organisms with high sleep drive and for exploring sleep strategies that provide resilience in extreme environments.

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.

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.

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.