Journal of Neurogenetics

Animals sleep more when they are sick. In C. elegans, stress-induced sleep (SIS) follows cellular injury such as exposure to ultraviolet (UV) light. The genetic regulators of SIS remain incompletely defined. Using a worm-picking robot, multi-well WorMotel imaging, and association analysis we performed a semi-automated screen of 941 whole-genome-sequenced Million Mutation Project (MMP) strains. We quantified behavioral activity and quiescence before and after ultraviolet (UV) radiation. We applied the Sequence Kernel Association Test (SKAT) to this behavioral data to prioritize 6,663 genes and observed significant enrichment of known SIS genetic regulators. Based on these results, we conducted a candidate validation screen for additional genes regulating SIS. We identified three genes (strd-1, egl-8, cla-1), mutations in which reproducibly influence SIS. Further exploration of these genes holds potential for enhancing our understanding of the molecular basis of SIS. These findings establish a pipeline for automated behavioral phenotyping coupled with gene-based association to accelerate studies of C. elegans neurogenetics.

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 using the flyPAD/optoPAD system. This method enables simultaneous measurement of feeding events across multiple arenas while allowing precise control of gustatory stimuli 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 consumption, nutrient preference, learning, and state-dependent modulation of feeding behaviors, and can be readily adapted for optogenetic manipulations and comparative choice assays.

Dominant missense mutations in ATP1A3, encoding a Na+, K+ ATPase -3 subunit, can cause Alternating Hemiplegia of Childhood (AHC), but how these mutations lead to AHC remains unclear. Here, we establish the first C. elegans AHC models by introducing AHC-causing ATP1A3 patient mutations (D801N, E815K, L839P, and G947R) into the orthologous gene, eat-6, using CRISPR/Cas9. Homozygous C. elegans AHC model animals have recessive developmental defects. Heterozygous AHC model animals have dominant defects in neuromuscular junction (NMJ) function that are inconsistent with haploinsufficiency and dominant sleep or arousal defects. Previous work in a Drosophila G755S AHC model found that loss of a K-dependent, Na/Ca{superscript 2} exchanger exacerbated neuronal defects. We introduced a loss-of-function allele of the orthologous C. elegans gene, ncx-4, into C. elegans AHC models; loss of ncx-4 function did not consistently alter C. elegans AHC model defects across alleles. Our results establish novel C. elegans models of AHC with robust phenotypes, demonstrate that AHC mutations disrupt NMJ function, and provide proof-of-concept for discovering cross-species modifiers of AHC-related phenotypes. Summary StatementWe report the first C. elegans models of Alternating Hemiplegia of Childhood. D801N, E815K, L839P, and G947R AHC model animals have recessive development defects and dominant neuromuscular defects.

Behavior arises from the complex interplay between an organisms nervous system, its genetic makeup, and the environment. High-resolution, high-throughput behavioral quantification is essential for dissecting biological function and the effects of genetic perturbation, but automated analysis remains challenging. Here, we present Autobehaver, an automated behavioral analysis pipeline based on a low-cost, high-throughput recording platform that captures videos of individual Drosophila. From each video, we extracted keypoints and used a custom Transformer to assign frame-wise behavior and orientation labels. We then converted these predictions into high-dimensional per-animal feature vectors and trained XGBoost ensembles to classify animals and identify the features that separated groups. By applying SHAP analysis to the classifier ensemble, we identified the behavioral features most informative for distinguishing groups of flies. We demonstrated the approach in several ways. First, we recovered known behavioral changes associated with heat-activated dTrpA1 activity in specific neural circuits. Second, we detected age-associated behavioral changes consistent with gradual impairment of locomotor and climbing ability. Finally, we used Autobehavers classifier ensemble to place animals with intermediate phenotypes along a behavioral axis and used feature-importance analysis to reveal the behavioral features underlying those intermediate states. Together, Autobehaver provides an interpretable framework for quantitative behavioral phenotyping and comparative analysis of complex genotypes.

The mosquito Aedes aegypti is an important vector of viral pathogens and serves as a model for other vector species. Pathogens are transmitted when a mosquito bites a host animal, but the neural circuits that control seeking and biting behavior are not known. Here, we detail methods and protocols for the manipulation of neural activity in the mosquito using optogenetics, a key technique to determine the causal relationship between neural circuits and behavior. These methods include rearing mosquitoes for optogenetics and three assays that are designed to measure different steps in the sequence of arousal, attraction, proboscis probing, and engorgement on the blood of host animals. These behaviors occur at different spatial scales and in response to different sensory stimuli. Each behavioral assay is outfitted with red (625 nm) LEDs for optogenetic activation. To detect arousal in response to olfactory stimuli, flight and walking are measured in all three assays. To assay attraction or landing, mosquitoes are presented with a heated blood meal in a large arena. Proboscis probing and engorgement are assayed with video resolution that enables measurement of appendages and abdomen size. The protocol describes machine vision models to enable high-resolution temporal quantification of behavior as well as endpoint measurements of feeding. These methods can be used to test the function of any population of neurons in mosquito biting behavior and can be extended to additional behaviors.

Long-term, automated tracking of group-housed social animals using RFID (radio frequency identification) is a promising approach in ethological neuroscience. However, low-frequency (LF) RFID, while long-established in the field, is constrained by its inherent low data rates, which lead to two critical limitations: (1) compromised spatiotemporal resolution, and (2) the inability to identify multiple tags (animals) simultaneously. To address these limitations, we developed eeeHive, a high-frequency (HF) RFID-based animal tracking system with a fully custom hardware architecture that enables high-speed, multiplexed antenna polling and concurrent multi-tag reading. The polling time per antenna in eeeHive was 5.9 ms, with an additional 8.2 ms read time per tag. We applied the system to track 24 mice for one week, and six common marmosets for seven weeks. The system successfully tracked individuals even within dense clusters, revealing complex behavioral traits characterized by spatial utilization, temporal dynamics, behavioral regularity, and inter-individual relationships. Additional tests with Japanese fire-bellied newts and Nile tilapia juveniles demonstrated comparable tracking performance in aquatic environments. Taken together, eeeHive overcomes the inherent limitations of conventional LF RFID, establishing a powerful HF RFID-based platform for fine-scale behavioral tracking of group-housed animals across terrestrial and aquatic species.

For animals to interact effectively with their environment, the brain must integrate sensory information and generate appropriate motor responses. Multiple neuronal circuits contribute to this process, and identifying their roles remains a central focus in neuroscience. The recently developed photoswitchable compound Carbadiazocine controls neuronal firing across species. It modulates larval zebrafish locomotion and alleviates neuropathic pain in rodents in a reversible, light-induced manner. Given its effects on both motor and somatosensory circuits, we investigated the impact of Carbadiazocine on sensorimotor behaviors. We focused on the optomotor response in zebrafish larvae and assessed its potential as a tool for circuit perturbation and behavioral analysis, for the first time combined with photopharmacology. We performed experiments in head-fixed and free-swimming larvae to assess their capacity to detect and follow the direction of optic flow, as well as to characterize swimming speed patterns and individual tail bout properties following administration of the two Carbadiazocine photoisomers. In both paradigms, treatment with the pre-illuminated compound led to a decrease in accuracy in responding to optic flow (correct turning percentage dropping from [~]95 % to [~]80 % in head-fixed experiments and correctness decreasing from [~]65 % to [~]20 % in free-swimming experiments). Speed analysis revealed an increased number and duration of fast movements with a decrease in number and duration of slow movements, even during periods without visual stimulation. Tail bout analysis further showed an increase in 15-30 Hz bout frequencies, corresponding to incomplete, irregular tail movements. All these effects were absent when the dark-relaxed compound was administered. Together, these findings deepen our understanding of sensorimotor transformations and lay the foundations to probe native neuronal circuits underlying behavior in diverse animal species using a wide dynamic range of photostimulation patterns.

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, Oct2R, Oct{beta}1R, Oct{beta}2R, Oct{beta}3R, and Oct-TyrR) showed extensive signal in the central nervous system. The same was found for the peripheral nervous system, with the exception of Oct{beta}2R, which showed pronounced expression in the somatic muscles. We also observed a previously undescribed role of Oct{beta}1R, Oct{beta}3R, 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.

At a mere 20 cells, the Caenorhabditis elegans intestine regulates metabolism, energy homeostasis, host defense, yolk production, and genetic aging, all while dynamically responding to its environment. How the intestine develops to carry out these disparate functions is unknown, and how cells differ along the length of the intestine is unclear. To address these questions, we performed single-cell RNA sequencing (scRNA-seq) on FACS-enriched intestinal cells from mixed-stage C. elegans embryos. The resulting single-cell transcriptomes of 974 cells organized into 13 clusters, suggesting a diversity of cell types and states. We used two post hoc approaches to ascribe identities to each cluster. First, genes with known developmental timing in early-, mid-, and late-stages were used to place clusters in time, and smiFISH microscopy was used to fine-tune the assignments. Second, the eight late-stage clusters were assessed for their region of origin. To assign these clusters to anatomical regions, we identified marker genes for each cluster and assessed their expression along the anterior-to-posterior length of the intestine using smiFISH microscopy. Genes associated with growth and cell division were expressed in early stages, whereas genes associated with immune responses and metabolism were expressed later. Genes associated with biotic responses and RNA metabolism were the most likely to vary across the intestines anterior-posterior axis. Finally, perturbation of anterior-localized intestinal transcripts more robustly affected intestinal function compared to central or posterior-localized genes. Overall, this research illustrates the intrinsic heterogeneity across the 20 cells of the embryonic intestine and sets the stage for future works aimed at understanding cell-specific intestinal responses to diet and the environment. ARTICLE SUMMARYWe investigate how the Caenorhabditis elegans intestine develops specialized functions on a spatiotemporal scale. We used single-cell RNA-sequencing to analyze embryonic intestinal cells and identify 13 distinct clusters. Combining gene expression analysis with microscopy, we assigned clusters to developmental stages and anatomical regions. Clusters associated with early intestine development express genes linked to growth and cell division, while later-stage clusters express genes involved in metabolism and immune responses. Genes varied across the intestines anterior-to-posterior axis, and disrupting anterior-specific genes produced stronger functional effects. These findings reveal previously unrecognized intestinal diversity and provide insight into how intestinal cells specialize during development.

Sleep is an essential behavioral state that evolved early in animals, possibly with the advent of the nervous system. The complexity of sleep neural networks varies significantly across phylogeny, yet common signaling molecules exist. Stress-induced sleep (SIS) of Caenorhabditis elegans is controlled by two sleep interneurons (ALA and RIS), within a 302-celled nervous system. Even in this simple framework, a complex array of signaling molecules is expressed. Here, we surveyed some of these genes for roles in SIS. These included neuropeptides, g-protein coupled receptors, a two-pored potassium channel, and glutamate signaling components. We found that multiple genes are required for sleep maintenance (i.e., amounts), and/or the precise timing of sleep initiation. In particular, we identified an important role for glutamate signaling. The conserved ionotropic glutamate receptor glr-5, regulates sleep maintenance and timing, and is required in a 3-celled circuit of interneurons connected by gap junctions and chemical synapses with RIS. This work suggests that numerous redundant and/or parallel mechanisms have evolved to modulate a simple sleep-regulating circuit in C. elegans, and we speculate that conserved pathways may play similar roles in animals with more complex systems.

Tau protein, the primary component in neurofibrillary tangles characteristic of Alzheimers Disease and related dementia disorders, normally regulates microtubule growth and stability. While tau dysfunction contributes to the progression of tauopathies, the role of microtubules in disease has remained unclear. Through forward genetic screening in Caenorhabditis elegans tauopathy models, we found multiple tubulin gene mutations that rescue tau-mediated neurodegeneration. Whole animal behavioral and in vitro biochemical assays were employed to characterize mutation-driven effects on neuron function, neurodegeneration, and effects on tubulin and tau proteins as well as microtubule function. Mutant tubulin genes were found to confer different levels of suppression correlating with the level of mutant gene expression. Mutant tubulins did not drastically alter total tau protein levels, tau phosphorylation or aggregation, however tau-induced neurodegeneration was rescued. The suppression of tau toxicity by tubulin gene mutations cannot be explained by changes in tau or tubulin expression, tau phosphorylation, or tau aggregation state. Rather the tubulin mutations appear to act by influencing global microtubule properties. In vitro experiments using C. elegans tubulin in semi-isolated and isolated contexts have indicated changes to microtubule properties without observable changes to tau-tubulin affinity. This work suggests that manipulation of microtubules can rescue tauopathy even when pathological tau species persist, supporting the importance of understanding microtubule contributions to disease progression and investigation into microtubule targeted gene therapy or small molecule approaches for tauopathy intervention.

How insects transmit food odour preferences acquired during the larval stage to their offspring is unknown. Bicyclus anynana butterfly larvae can learn to prefer a banana-smelling odour, isoamyl acetate (IAA), via feeding on coated leaves, or simply via haemolymph transfusions from an IAA-fed animal, and transmit this preference to their naive offspring. Here we explore how larvae respond to different concentrations of IAA using olfaction choice tests, and how injections of different concentrations of IAA directly into the haemolymph impact odour learning and transmission of learned preferences. We find that naive larvae showed a slight preference towards low concentrations of IAA, and a slight avoidance towards higher concentrations. Injections of IAA at low concentrations directly into the haemolymph led to an increase in preference for IAA, whereas higher concentrations led to an increase in avoidance. Naive offspring inherited the odour preferences of their parents. Finally, injections of IAA at different concentrations into embryos did not alter choices made by hatched larvae. We establish that the same molecule (IAA) can illicit both a preference as well as an aversive reaction when directly injected into the haemolymph, but IAA is not directly implicated in intergenerational inheritance.

Chemical risk assessment for insects relies largely on mortality endpoints in a few model species, limiting detection of ecologically relevant sublethal effects and cross-taxon comparisons. Behaviour is a sensitive indicator of neurotoxic stress, but scalable, standardised measurement has remained challenging. We present BEEhaviourLab, a low-cost, automated platform for high-throughput behavioural phenotyping. The system integrates parallel video and audio recording with computer vision tracking, acoustic classification, experimental control, and automated analyses, enabling long-duration experiments across many replicates and species. A single lightweight object-detection model tracks multiple untagged insects simultaneously, including individuals from different taxa (e.g., bees and hoverflies). Using this platform, we quantify species-specific circadian activity patterns and assess toxicity of the widely used veterinary pesticide moxidectin in the bumble bee (Bombus terrestris). Acute contact exposure caused dose-dependent reductions in locomotion and buzzing at sublethal concentrations, with acoustic measures more sensitive than video. These results demonstrate that scalable, multimodal behavioural phenotyping can detect biologically meaningful neurotoxic disruption well below lethal thresholds, providing a practical path to integrate sublethal endpoints into chemical risk assessment.

Sleep-wake regulation arises from the interaction between homeostatic sleep pressure and circadian timing, yet current assessments evaluate these processes independently and fail to capture their dynamic modulation by environmental pressures. This limitation is particularly relevant in adolescents with attention-deficit/hyperactivity disorder (ADHD), who are at increased risk of circadian delay and sleep disruption. Here, we combined month-long wearable-based physiological monitoring with ecological behavioral assessments in adolescents with ADHD to characterize circadian and homeostatic processes dynamically in real-world settings. Using continuous skin temperature recordings, we derived individualized and day-specific estimates of circadian phase through hierarchical modelling, and integrated these measures with actigraphy-based sleep estimates and daily assessments of neurocognitive functioning and functional impairment. Temperature-derived circadian phase correlated with questionnaire-based chronotype but more accurately predicted sleep patterns. Delayed circadian phase was associated with later sleep onset and greater weekday-weekend variability. Importantly, circadian phase exhibited significant day-to-day fluctuations, particularly in individuals with delayed phase, reflecting interactions with environmental constraints. Sleep latency was jointly determined by homeostatic sleep pressure and day-specific circadian phase, with combined models outperforming either process alone. Crucially, both sleep deprivation and day-specific circadian misalignment independently predicted fluctuations in ADHD symptom severity, perceived stress, and neurocognitive impulsivity. In contrast, mean circadian phase alone did not explain behavioral variability. These findings demonstrate that circadian regulation is a dynamic, environmentally sensitive process rather than a fixed trait. Wearable-based estimation of circadian phase provides a scalable approach to capture these dynamics and may enable personalized interventions targeting sleep and circadian dysregulation.

Alzheimers disease (AD) is a devastating neurodegenerative disorder characterized by memory loss and a decline in cognitive function. Hallmarks of AD include an age-dependent accumulation of toxic amyloid beta (A{beta}) 42 in the brain, energy dyshomeostasis caused by mitochondrial dysfunction, and iron overload. However, the role of iron overload and mitochondrial dysfunction in AD pathology is unknown and their precise relationship with A{beta} 42 toxicity in AD pathology is unclear. C. elegans provide a powerful model system to untangle and clarify these relationships. In this study, we quantify the temperature-dependence of iron toxicity (16, 20 and 25C) in neurons and muscle of C. elegans that overexpress A{beta} 42. We found that A{beta} 42, regardless of the cell-type expression, caused accelerated paralysis compared to age-matched WT worms with the greatest degree of paralysis observed at an elevated temperature (25C). Moreover, the combination of iron toxicity and A{beta} 42 results in an enhanced paralytic phenotype at 16C. Thus, iron exposure potentiates A{beta} toxicity observed at low temperatures. Iron toxicity stimulated both maximum (State 3) and leak (State 4) respiration in WT and A{beta} 42 worms. A{beta} 42 worms also exhibited increased leak respiration at baseline that was further exacerbated by iron toxicity. Iron burden and sensitivity increased A{beta} 42 peptide toxicity. A{beta} 42 worms exhibited reduced levels of Ca, Zn, Mn, and K. Overall, our results suggest that iron potentiates A{beta} toxicity at low temperature and enhances A{beta} peptide mediated mitochondrial bioenergetic dysfunction in C. elegans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/714217v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9eaf46org.highwire.dtl.DTLVardef@542eforg.highwire.dtl.DTLVardef@16d9678org.highwire.dtl.DTLVardef@1b1b16d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITemperature stress modulates the synergetic interactions of iron toxicity and A{beta} 42 pathology C_LIO_LIIron sensitivity drives increased cell-type specific A{beta} 42 pathology C_LIO_LIEnergy dyshomeostasis via impaired mitochondrial function and increased proton leak contributes to iron- and A{beta}-induced pathology C_LI

The role of vision in the behavior of blood-feeding mosquitoes has remained largely overlooked, particularly in species of the Anopheles genus, despite their significant impact on global health. While the importance of olfactory and thermal cues in host-seeking is well established, the contribution of visual stimuli to mating and feeding behavior remains far less understood. In particular, Anopheles mosquitoes exhibit complex swarming behavior that depends on visual input, suggesting an underexplored avenue of research with direct implications for vector control. This study introduces a genetically modified mosquito line lacking the enzyme Tan, a hydrolase involved in both dopamine and histamine metabolism, to investigate the behavioral relevance of visual cues in Anopheles. Through a combination of behavioral assays and controlled laboratory experiments, the impact of visual disruption on attraction to UV-B light, host-seeking, and blood-feeding success was assessed. The findings demonstrate a reduced light-dependent attraction in both Anopheles males and females, suggesting an impairment in visual processing or a related behavioral response. This observation has implications for reproductive success and potential adaptation to anthropogenic environments with artificial light. By leveraging this novel knockout model, the study offers new tools and perspectives to better understand how vision shapes mosquito behavior and how this knowledge could be harnessed in the development of next-generation vector control strategies.

Bimodal gene expression systems have played a major role in uncovering the function of genes, cells and organ systems during and after development. Employed initially in model systems such as flies and mice, advances in gene technology have vastly expanded the number of species in which these systems can be deployed. One of their limitations is the challenge of imposing temporal expression control. Here, we report the incorporation of temperature-sensitive intein modules with different temperature profiles into QF2, the transcription factor anchoring the Q-system widely used in Drosophila and introduced recently into several insect species and the zebrafish D. rerio. Intein removal from temperature-sensitive QF2_INTts activators in Drosophila larvae and adult flies raised under permissive conditions (18{degrees}C to 25{degrees}C) renders QF2 active and drives strong expression of QUAS reporters. In contrast, raising Drosophila at restrictive temperatures (23{degrees}C to 30{degrees}C) keeps QF2_INTts in a non-functional state unable to bind DNA and therefore, keeping QUAS reporters inactive. We further show that reporter expression can be turned on and off both during development and in flies by changing temperature conditions between restrictive to permissive. Finally, QF2_INTts animals carrying QUAS-Kir2.1 encoding the inward rectifying potassium channel raised under restrictive conditions are fully viable, while raised at or moved as adults to permissive temperature results in embryonic lethality or leads to paralysis. Thus, the intein-based Q-system renders the difficult to employ drug-dependent suppressor QS unnecessary, greatly facilitating temporal regulation of gene expression. Having several advantages over the GAL4 system, the Q system has gained broad acceptance in other insect species and the zebrafish D. rerio, and thus QF2_INTts drivers can be implemented in many other organisms, including disease-transmitting mosquitoes and poikilotherm vertebrate animals much more closely related to humans, to study gene and cell function at any time during or after development.

Zebrafish have been used a prominent model for high-throughput phenotypic screens of candidate risk gene mutations for several disorders. This also includes models for attention deficit/hyperactivity disorder (ADHD). Traditional behavioural tests, such as the forced light/dark assay, concentrate on basic locomotion measures. However, recently developed visually-driven locomotion assays, for example closed-loop systems using virtual reality, have allowed extraction of richer data on animal locomotion and decision-making under different sensory stimuli. Here, we have used such a system to assess the behaviour in adgrl3.1 mutant fish, an established model for ADHD. Our results show that mutants exhibit a higher baseline excitability and a lower threshold for initiating motor events, demonstrating that collecting behavioural responses in an interactive environment enables a more precise characterisation of ADHD-relevant phenotypes associated with adgrl3.1 disruption. More generally, we establish a scalable translational platform to screen gene-function relationships and possible therapeutic interventions, not only for ADHD but multiple neurodevelopmental disorders.

Spatial navigation could theoretically serve as an early neurobehavioral marker of Alzheimers disease risk, yet technological limitations have hindered its widespread adoption. We leveraged breakthroughs in technology to create a custom smartphone application to compare real-world spatial memory with lab-based measures. Specifically, we compared performance across two established lab-based tasks, judgments of relative direction (JRD) and map drawing, and our novel app-based, in situ pointing task administered in a familiar large-scale, real-world environment. Young adults completed both laboratory and mobile navigation tasks, allowing within-subject comparisons across modalities. JRD performance strongly correlated with map drawing performance. In contrast, App-based pointing showed lower error and reduced inter-individual variability relative to JRD performance, but weak correlations with lab-based measures. We also developed a novel analytical technique in which we transformed the app-based pointing into a relational, JRD-like metric, and we observed strong correlations and correlated patterns of errors across all tasks. Thus, real-world, app-based pointing captures stable directional performance (e.g., as indexed by the lower errors and lower variability relative to the JRD Task) and, when expressed in a common framework, correlates with laboratory measures of spatial memory, thus suggesting that these tasks tap into partially overlapping cognitive representations. These results provide a pivotal advancement to our understanding of both shared and unique variance across spatial memory paradigms, and support the use and further development of mobile navigation tools as scalable complements to lab-based assessments for studying spatial cognition and its decline in preclinical and clinical stages of Alzheimers disease. HighlightsO_LISpatial memory is a core cognitive function and is impaired in Alzheimers disease C_LIO_LITesting memory in large-scale, real-world environments enhances ecological validity C_LIO_LIWe compared performance of our novel real-world measure with lab measures C_LIO_LIWe observed strong correlations between the lab-based measures C_LIO_LIWe observed shared and unique variance between lab- and real-world measures C_LI

The ability of flying insects to locate distant food and mates by tracking odor plumes through turbulent and unsteady flow represents a remarkable feat of sensorimotor integration. Successful navigation requires not only extracting a reliable directional estimate from an intermittent olfactory signal, but also contending with the challenging dynamics of variable winds. While prior work has established that insects integrate the history of odor encounters to shape search decisions, whether they also retain a working memory of recently experienced wind conditions has remained unknown. Here, we use optogenetics combined with controlled wind perturbations in a free-flight wind tunnel to investigate how wind history modulates the olfactory search behavior of Drosophila melanogaster. By introducing lateral "gust" flow via auxiliary fans and independently delivering olfactory stimuli, we show that the wind experienced during an olfactory stimulus shapes both the immediate surge response and the subsequent spatial search. Flies that received an olfactory stimulus while being displaced by a crosswind gust were significantly more likely to return to the gust zone during the post-stimulus search phase compared to flies that received the same odor cue in steady laminar flow. Meanwhile, surge responses and course directions exhibited during search indicate that moment-to-moment flight kinematics may be driven more by instantaneous flow. These results reveal that wind experience is tracked in addition to olfactory experience, and provide evidence that Drosophila maintain a short-term working memory of ambient wind conditions to guide olfactory navigation.