Andreas S. Thum

Animals can adapt their behavioral responses to environmental cues by learning from experience. This ability relies on the formation and recall of memories that are shaped by beneficial or detrimental consequences and regulated by the dopaminergic system, which is highly conserved across insect species. In the Drosophila melanogaster larva, eight of total ∼120 dopaminergic neurons (DANs) innervate the mushroom body (MB), a key center for associative memory. This subset of DANs can be anatomically grouped into two clusters of four cells: the primary protocerebral anterior medial (pPAM) cluster, associated with reward signaling, and the dorsolateral 1 (DL1) cluster, associated with punishment. Such a functional dichotomy is observed in larval and adult Drosophila and reflects a fundamental organizational principle of reinforcement learning across invertebrate and even vertebrate species. Aversive reinforcement through high-salt exposure is encoded within the DL1 cluster in a combinatorial and heterogeneous manner, critically involving two neurons, DAN-f1 and DAN-g1. Using temporally precise optogenetic activation and inhibition during olfactory conditioning, we show that these neurons can modulate memory strength and valence. Their effects are most often consistent and predictable, enabling accurate computational modeling of DAN-driven teaching signals. By manipulating the intrinsic physiology of DAN-f1 and DAN-g1 and altering the valence of gustatory input, we are beginning to understand at the single-cell level how dopaminergic activity is systematically adjusted to control memory formation.

This study provides a comprehensive ultrastructural analysis of the pharyngeal sensory system in Drosophila melanogaster larvae, focusing on the four major pharyngeal sense organs: the ventral pharyngeal sensilla (VPS), dorsal pharyngeal sensilla (DPS), dorsal pharyngeal organ (DPO), and posterior pharyngeal sensilla (PPS). Our analysis revealed 15 sensilla across these organs, comprising four mechanosensory, nine chemosensory, and two dual-function sensilla. We identified 35 Type I neurons (6 mechanosensory and 29 chemosensory) and 6 Type II neurons with putative chemosensory functions. Additional sensory structures, including papilla sensilla and chordotonal organs in the cephalopharyngeal region, were characterized. This detailed mapping and classification of pharyngeal sensory structures completes their structural characterization and provides a foundation for future anatomical and functional studies of sensory perception in insects. This work represents a significant step toward a complete analysis of the larval sensory system, providing new opportunities for investigating how an organism processes sensory information to navigate and interact with its environment.

This study provides a comprehensive ultrastructural analysis of the pharyngeal sensory system in Drosophila melanogaster larvae, focusing on the four major pharyngeal sense organs: the ventral pharyngeal sensilla (VPS), dorsal pharyngeal sensilla (DPS), dorsal pharyngeal organ (DPO), and posterior pharyngeal sensilla (PPS). Our analysis revealed 15 sensilla across these organs, comprising four mechanosensory, nine chemosensory, and two dual-function sensilla. We identified 35 Type I neurons (six mechanosensory and 29 chemosensory) and six Type II neurons with putative chemosensory functions. Additional sensory structures, including papilla sensilla and chordotonal organs in the cephalopharyngeal region, were characterized. This detailed mapping and classification of pharyngeal sensory structures completes their structural characterization and provides a foundation for future anatomical and functional studies of sensory perception in insects. This work represents a significant step towards a complete analysis of the larval sensory system, providing new opportunities for investigating how an organism processes sensory information to navigate and interact with its environment.

Octopamine is involved in a variety of physiological and behavioral mechanisms throughout the Drosophila melanogaster life cycle. Octopaminergic neurons in both the central and the peripheral nervous system target a multitude of neurons and even non-neuronal tissues, making it challenging to analyze individual mechanisms of octopamine function. One approach to deconstructing this complex system is to examine the postsynaptic components of signal transmission, the six distinct G-protein-coupled octopamine receptors. Here, we compiled and validated a complete set of potential mutant lines of all known octopamine receptors (Oamb, Octα2R, Octβ1R, Octβ2R, Octβ3R, and Oct-TyrR) that were generated using the same genetic tool: the recently established Trojan Exon system. It integrates the Gal4/UAS binary expression strategy while simultaneously impairing receptor function. We generated a comprehensive anatomical map of receptor expression in the larva and, at the same time, analyzed the function of individual octopamine receptors during larval development, chemosensory perception, and locomotion. All octopamine receptors are expressed in the central and peripheral nervous system. Octβ2R stands out with a pronounced expression in the somatic muscles. We also observed a previously undescribed role of Octβ1R, Octβ3R, and Oct-TyrR in larval hatching and in the survival of larvae and pupae. Combined with tissue- and stage-specific gene expression data, this validated set of transgenic lines provides a basis for further functional studies of the octopaminergic system.

Animals can adapt their behavioral responses to environmental cues by learning from experience. This ability relies on the formation and recall of memories that are shaped by beneficial or detrimental consequences and regulated by the dopaminergic system, which is highly conserved across insect species. In the Drosophila melanogaster larva, eight of total ∼120 dopaminergic neurons (DANs) innervate the mushroom body (MB), a key center for associative memory. This subset of DANs can be anatomically grouped into two clusters of four cells: the primary protocerebral anterior medial (pPAM) cluster, associated with reward signaling, and the dorsolateral 1 (DL1) cluster, associated with punishment. Such a functional dichotomy is observed in larval and adult Drosophila and reflects a fundamental organizational principle of reinforcement learning across invertebrate and even vertebrate species. Aversive reinforcement through high-salt exposure is encoded within the DL1 cluster in a combinatorial and heterogeneous manner, critically involving two neurons, DAN-f1 and DAN-g1. Using temporally precise optogenetic activation and inhibition during olfactory conditioning, we show that these neurons can modulate memory strength and valence. Their effects are most often consistent and predictable, enabling accurate computational modeling of DAN-driven teaching signals. By manipulating the intrinsic physiology of DAN-f1 and DAN-g1 and altering the valence of gustatory input, we are beginning to understand at the single-cell level how dopaminergic activity is systematically adjusted to control memory formation.

The apical extracellular matrix (aECM) serves as a dynamic scaffold, orchestrating tissue growth, cellular communication, and wound repair, and thus functions as a powerful biomaterial. In this study, we explored the intricate three-dimensional landscape of the Drosophila larval aECM using advanced 3D Volume electron microscopy. This technique opens a window into understanding and mimicking the diversity of aECM surfaces and architectures of the exoskeletal cuticles. We reconstructed the striking forms of denticles, hairs, and sensory organs, including double sensilla and Keil’s organs, at the epidermal surface. Our reconstructions brought the larval tracheal system and the sponge-like felt chamber (Filzkörper) of the spiracles into sharp relief, revealing a labyrinth of tiny cavities surrounding a central tube that extends from the posterior opening to the tracheal lumen. When examining the ultrastructure of transport-active anal pad cells, we discovered an inverted pattern of cuticular pore-canal structures, which supports their suggested role in osmoregulation across the aECM. Our findings further suggest that ordinary epidermal cells weave an intricate network of pore canals, enabling the orchestration of material distribution throughout the entire aECM. Moreover, we reconstructed tendon cells and chordotonal organs that form a branching network of numerous filamentous intracuticular fibers that weave through the aECM, linking the exoskeleton to muscles and sensory organs. By thoroughly mapping the intricate ultrastructural features of the aECM, this study opens new avenues for exploring how its protective, mechanical, and sensory roles intertwine, inspiring the next generation of tailor-made biomimetic materials.

Octopamine is involved in a variety of different physiological and behavioral mecha-nisms in Drosophila melanogaster . Throughout the life cycle of the fruit fly, from the larva to the adult, octopaminergic neurons in both the central and the peripheral nerv-ous system target a multitude of neurons and even non-neuronal tissues, making it challenging to analyze individual mechanisms of octopamine function. One approach to deconstructing this complex system is to examine the postsynaptic components of signal transmission. In Drosophila , octopamine interacts with six distinct G-protein-coupled receptors. For some of these receptors, expression maps and functional im-plications have been described. In contrast, other receptors have been neglected, partly due to the lack of suitable genetic tools. Here, for the first time, we compiled a complete set of mutant lines of all known octopamine receptors, all generated using the same genetic tool, the recently established Trojan Exon system. It integrates the Gal4/UAS binary expression strategy while simultaneously impairing receptor func-tion. This enabled us to generate a comprehensive anatomical map of receptor ex-pression in the larva and, at the same time, analyze the function of individual octopa-mine receptors during larval development, chemosensory perception and locomotion. All octopamine receptors (Oamb, Octα2R, Octβ1R, Octβ2R, Octβ3R, and Oct-TyrR) showed extensive signal in the central nervous system. The same was found for the peripheral nervous system, with the exception of Octβ2R, which showed pronounced expression in the somatic muscles. We also observed a previously undescribed role of Octβ1R, Octβ3R, and Oct-TyrR in larval hatching and in the survival of larvae and pupae. Molecular evaluation of the Trojan Exon octopamine lines supports our analy-sis. In addition, we combined the experimental results with gene expression data from the different development stages of Drosophila melanogaster and from different tis-sues and cell populations throughout the body. Overall, we compiled, analyzed and validated a complete set of octopamine lines which, together with gene expression analysis, provides a basis for further functional studies on the larval octopaminergic system.

The catecholamines octopamine and tyramine undoubtedly have a major impact on the life of an insect. A wide range of physiological processes and behaviors are regulated by these neurotransmitters/hormones. Octopamine and tyramine act homologous to the adrenergic system of vertebrates, primarily adapting the organism to the given situation, by switching between the states of alertness and rest. Interestingly, higher brain functions like learning and memory are also regulated by octopamine and tyramine. About 30 y ago, initial work in Drosophila demonstrated that dopaminergic neurons signal punishment, while octopaminergic neurons signal reward during olfactory associative learning and memory. In the meantime, however, it has become clear that distinct types of dopaminergic neurons convey both reward and punishment signals to the mushroom bodies, a central brain region responsible for the formation and storage of associative memories. Although some conflicting data remain, these findings challenge the previously established model of functional segregation and may limit the proposed role of octopamine neurons in mediating reinforcing information during memory formation. We have therefore re-examined the role of octopamine in learning and memory in Drosophila larvae. Our findings suggest that optogenetic activation of octopaminergic neurons is sufficient to drive appetitive and aversive memory formation. Further, through a combination of Ca ²⁺ imaging, anatomical studies, and loss-of-function behavioral approaches, we demonstrate that octopamine signaling plays a crucial role in larval learning by modulating dopaminergic neurons across distinct cell clusters to orchestrate memory processes.

Orientation and navigation are essential features of animals living in changing environments. Typically, animals integrate a variety of allothetic and idiothetic cues to achieve their navigational goals. Allothetic cues, such as visual or chemical landmarks from the environment, provide an external frame of reference. In contrast, idiothetic cues are based on internal proprioceptive feedback and internal copies of motor commands. When Drosophila larvae are exposed briefly to a Teflon container holding a food stimulus, they show a characteristic behavior as soon as the container is removed: they briefly crawl away from the detected resource, remain in its vicinity and then return to the area where they experienced the earlier stimulus. We quantified this behavior with respect to the chemosensory nature of the stimulus, starvation time and genetic background of the larvae. We conclude that this behavior represents a centered local search. Furthermore, we exclude various external stimuli (vision and taste), which suggests that possibly idiothetic as opposed to allothetic cues have a stronger influence on the larval local search behavior. In the long term, this behavioral description will enable us to gain insights into the comparability of larval foraging strategies. We also want to investigate whether, despite the simpler organization of the larval brain and the alleged lack of a central complex, a brain region that is important for orientation and navigation in adult Drosophila and other insects, there are common solutions for the brain circuits underlying search behavior.

Summary: Sleep is a continuous behavior across the lifespan, yet its features and functions evolve markedly with development ^1–4 . In Drosophila melanogaster, as in mammals, early life sleep differs from mature sleep ^5,6 , but it is unknown whether disparate sleep regulatory mechanisms underlie these changes. Here, we identify distinct populations of octopaminergic (OA) neurons that promote arousal in larval and adult flies, thus revealing a developmental switch in sleep-wake circuit architecture. Of eight OA neurons present in the sub-esophageal zone (SEZ) of the nervous system at both life stages, dedicated, non-overlapping subsets drive arousal in larvae versus adults. Morphologic and connectomic analyses show that larval OA arousal neurons project primarily to the ventral nerve cord and lack substantial sensory input, suggesting a circuit logic optimized for internally driven arousal during early development. In contrast, adult OA arousal neurons target higher brain regions involved in cognition and receive rich multimodal sensory input, supporting wakefulness in response to environmental cues. These findings highlight a developmental transition in arousal circuitry that mirrors changing ecological demands, with juvenile systems organized to prioritize growth and feeding, insulated from sensory disturbance, and mature systems supporting sensory-guided behavior. Our results support a model of sleep regulation as a developmentally dynamic process, in which shared neuromodulators like OA operate through distinct cellular substrates tailored to life stage– specific behavioral priorities.