Quantifying feeding behavior with high temporal and spatial precision is critical for understanding how internal state, sensory cues, and neural activity shape food intake and dietary choice. Here, we describe a detailed protocol for performing consumption and dietary choice assays in Drosophila melanogaster using the flyPAD/optoPAD system. This method enables simultaneous measurement of feeding events across multiple arenas while allowing precise control of sensory and optogenetic stimulation. We provide step-by-step instructions for assay food preparation, flyPAD arena setup, data acquisition, and downstream data organization with suggested analyses. This approach is suitable for studying nutrient preference, learning, and state-dependent modulation of feeding behavior, and can be readily adapted for optogenetic manipulations and comparative choice assays. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, R35GM147504
Author summary For more than a century, neuroscience has focused on connections between neurons via synapses as the main basis of brain function. In this study, we explore an additional and less understood layer of neuronal communication: chemical signaling, mediated by small diffusible molecules called neuropeptides. Neuropeptides are conserved across organisms and also present in humans and do not require synaptic wiring and instead can act more like radio broadcast messages, reaching many cells at once, and activating only those with the right receptor. We chose the simple freshwater animal Hydra to study this form of communication because its nervous system is small, accessible, and can be studied with whole-body imaging and genetic analysis. By analyzing its genome and gene expression, we identified a rich set of neuropeptides and their matching receptors, revealing an extensive chemical communication network. We found that different neuron types use distinct combinations of these signals, suggesting organized communication pathways despite the absence of a centralized brain. By modeling this network, we show that such chemical signaling alone can support stable patterns of activity, similar to the neural networks thought to underlie behavior and memory in more complex brains. Our findings suggest that chemical communication is an ancient and fundamental principle of nervous system organization, providing a complementary framework to synaptic wiring for understanding how brains generate behavior.
Following the evolution of internal fertilisation, the female reproductive tract became the site of reproductive interactions setting the stage for cooperation and conflict over reproductive outcomes. After mating, females undergo a series of changes in morphology, physiology and behaviour. These postmating female responses are critical to fertility. Traumatic insemination observed in the common bedbug (Cimex lectularius) presents an unusual scenario under which postmating responses unfold. Bedbugs have evolved a novel organ, the spermalege, that is the site of insemination and initial ejaculate x female interactions. As the female reproductive tract does not take receipt of the ejaculate until several hours after mating, bedbugs also provide a unique opportunity to decouple female reproductive functions involved in mating from those relating to insemination. Here we show the novel mesospermalege organ has acquired many functions normally associated with the reproductive tract in other species, including immunogenic and ejaculate processing functions, as well as expression of male seminal fluid genes. This pattern of intersexual molecular dynamics previously shown in Drosophila suggests a more common pattern of shared expression of reproductive genes. We also show that the postmating response in the female reproductive tract is delayed, coinciding with the movement of sperm through the female, clearly showing that the postmating response has evolved in response to sperm receipt rather than being an innate function of the tissue. Our results provide insights into the evolution of novel reproductive traits and female postmating physiology in an economically important global pest. ### Competing Interest Statement The authors have declared no competing interest. BMFTR and the Free State of Saxony under the Excellence Strategy of Federal Government and the Länder National Science Foundation, PRFB-2208973
Feeding behavior requires the integration of environmental cues, metabolic signals, and internal states through neuromodulatory networks, yet the role of specific neuromodulatory neurons and how they are coordinated in controlling feeding remains unclear. Using automated feed–tracking and optogenetics in Drosophila melanogaster , we found that activation of two octopaminergic neurons (VPM3/4) increases food consumption. Both VPM neurons form direct synapses with a mushroom–body output neuron MBON11, which, through opto–activation and opto–inhibition, can exert bidirectional control over food intake, with sexually dimorphic effects. Epistasis experiments demonstrated that octopamine–driven feeding requires functional MBON11 output. Additionally, we found that the dopaminergic PPL101 neurons, which also synapse with MBON11, are required for hunger–driven feeding. Ethomic analysis contextualized with natural hunger and satiety provided a holistic view of how different neuron types linked to the mushroom–body influence feeding behaviors. This analysis revealed that MBON11 interventions best recapitulate natural hunger–satiety transitions. These findings revealed a circuit where two neuromodulatory neuron types with distinct unidirectional feeding effects—octopaminergic VPM3/4 (instructive but not required) and dopaminergic PPL101 (required but not instructive)—converge onto MBON11, a neuron whose activity is both required for and instructive of hunger-related feeding. This circuit arrangement may represent an architecture for integrating multiple motivational signals in feeding regulation. ### Competing Interest Statement The authors have declared no competing interest. Ministry of Education, Singapore, MOE2017-T2-1-089, MOE2019-T2-1-133, FY2022-MOET1-0001, MOE-T2EP30222-0018 A*STAR Joint Council Office, https://ror.org/01pz9tv63, 1231AFG030, 1431AFG120 National Medical Research Council (NMRC), MOH-OFYIRG20nov-0051 Yong Loo Lin School of Medicine (NUS Medicine) scholarship A*STAR Scientific Scholars Fund
flypapers